US20220267754A1 - Vector-based mutagenesis system - Google Patents

Abstract

Strategies, reagents, methods, and systems for modulating the mutation rate in cells are provided herein. The strategies, reagents, methods, and systems are broadly applicable for the modulation of mutation rates in cells where high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products.

Description

RELATED APPLICATIONS
• The present application claims the benefit under 35 U.S.C. § 119(e) to U.S. provisional patent applications. U.S. Ser. No. 62/149,378, filed Apr. 17, 2015, and U.S. Ser. No. 62/272,035, filed Dec. 28, 2015 the entire contents of each of which are incorporated herein by reference.
GOVERNMENT SUPPORT
• This invention was made with Government support under grant HR0011-11-2-0003, awarded by Defense Advanced Research Projects Agency (DARPA), and under grant N66001-12-C-4207 awarded by the Space and Naval Warfare Systems Command (SPAWAR). The Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
• The random mutagenesis of DNA provides the genetic diversity that fuels evolution both in nature and the laboratory. Methods to enhance unbiased, random mutagenesis in cells offer major advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency, and narrow mutational spectra.
SUMMARY OF THE INVENTION
• Some aspects of this disclosure provide strategies, reagents, compositions (e.g., vectors), kits, methods, and systems for modulating the mutation rate in cells. The strategies, reagents, compositions, kits, methods, and systems are broadly applicable for the modulation of mutation rates in cells in any context in which high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products. While some uses and applications are described in detail herein, additional suitable uses of the strategies, reagents, compositions, kits, methods, and systems provided herein will be apparent to those of skill in the art based on the present disclosure.
• Phage-assisted continuous evolution (PACE) can serve as a rapid, high-throughput method to evolve genes of interest. One advantage of the PACE technology is that both the time and human effort required to evolve a gene of interest are dramatically decreased as compared to conventional iterative evolution methods. The general concept of PACE technology has been described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference. During PACE, a phage vector carrying a gene encoding a gene of interest replicates in a flow of host cells through a fixed-volume vessel (a “lagoon”). For example, in some embodiments of PACE described herein, a population of bacteriophage vectors replicates in a continuous flow of bacterial host cells through the lagoon, wherein the flow rate of the host cells is adjusted so that the average time a host cell remains in the lagoon is shorter than the average time required for host cell division, but longer than the average life cycle of the vector, e.g., shorter than the average M13 bacteriophage life cycle. As a result, the population of vectors replicating in the lagoon can be varied by inducing mutations, and then enriching the population for desired variants by applying selective pressure, while the host cells do not effectively replicate in the lagoon.
• The rate at which host cells mutate a gene of interest during a directed evolution experiment can affect the time required to evolve a gene of interest to a desired state, and may even limit the mutations that can be achieved during a standard PACE experiment. While conventional methods of mutagenesis are effective, in some circumstances, higher mutation rates are desirable. Some aspects of this disclosure provide potent, inducible, broad-spectrum, and expression construct-based (vector-based) mutagenesis systems for use in bacterial cells, e.g.,E. colicells. These recombinant expression constructs, also referred to as mutagenesis constructs or, if they are in the form of a plasmid, as mutagenesis plasmids, can enhance mutation rates by over 300,000-fold as compared to basal levels when fully induced, surpassing the mutational efficiency and spectra of the most widely used in vivo and in vitro mutagenesis methods. In some embodiments, the mutation rate can be modulated via inducible expression of mutagenic gene products from the expression constructs provided herein, which are useful for modulating the mutation rates as desired, e.g., during different phases of a directed evolution experiment.
• Some aspects of this disclosure demonstrate the usefulness of the mutagenesis systems provided herein in a directed evolution context, e.g., for evolving biomolecules with novel or changed properties. The improved mutation rates are demonstrated, for example, by the data provided in the Examples, showing that novel antibiotic resistance can be evolved in wild-typeE. coliin less than 24 hour using the mutagenesis expression constructs, thus outperforming all known methods for inducing mutations in host cells, including, for example, chemical mutagens, UV light, and the mutator strain XL1-Red under similar conditions. The mutagenesis systems provided herein also allowed for the rapid continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in less than 10 hours without requiring any evolutionary stepping-stones or initial mutational drift, in contrast to previously described mutagenesis systems with lower mutation rates. The mutagenesis systems, methods, and kits provided herein can be applied, for example, to the high-frequency, broad-spectrum mutagenesis of chromosomal, episomal, and viral nucleic acids in vivo, and are applicable, inter alia, to a variety of cells, e.g., bacterial, yeast, and eukaryotic cells, and a variety of applications, e.g., both bacterial or bacteriophage-mediated laboratory evolution platforms. Those of skill in the art will understand that these examples are provided to illustrate some non-limiting, possible applications of the mutagenesis systems and expression constructs provided herein, and that the present disclosure embraces additional applications and is not limit in this respect.
• In some embodiments, a plurality of nucleic acid sequences encoding a gene product that increases the mutation rate in a cell are employed, e.g., combinations of such gene products as shown in Table 2 orFIG. 7. In some embodiments, combinations of two or more nucleic acid sequences encoding different gene products that increases the mutation rate in a cell may be included in a single expression construct, e.g., in a multi-cistronic construct in which a single promoter, e.g., an inducible promoter as described herein, drives expression of all or a plurality of the different encoding sequences, or in an expression construct comprising two or more promoters, each driving expression of at least one of the encoding sequences. In some embodiments, a combination of different encoding sequences may be provided on different expression constructs, each of which carrying at least one of the different encoding sequences. Suitable configurations or monocistronic, multicistronic, and multi-vector expression constructs for expression of the combinations of mutagenesis-inducing gene products in cells will be apparent to those of skill in the art in view of the present disclosure.
• In some embodiments, the gene product that disrupts a proof-reading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product. In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product.
• In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product. In some embodiments, the recA gene product is a recA730 gene product. In some embodiments, the polB gene product is a polB(D156A) gene product. In some embodiments, the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product. In some embodiments, the mutS gene product is a mutS538, mutS503, or mutSΔN gene product. In some embodiments, the mutL gene product is a mutL705, mutL713, mutL(R261H), or mutL(K307A) gene product. In some embodiments, the mutH gene product is a mutH(E56A), mutH(K79E), or mutH(K 116E) gene product. In some embodiments, the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product. In some embodiments, the AID gene product is an AID (7), AID (7.3), AID (7.3.5), AID (7.3.3), AID (7.3.1), or AID (7.3.2) gene product. In some embodiments, the APOBEC gene product is an APOBEC 1 gene product. In some embodiments, the CDA gene product is a CDA1 gene product. In some embodiments, the MAG gene product is a MAG1 gene product. In some embodiments, the AAG gene product is an AAG(Y127I-H136L) or Δ80-AAG(Y127I-H136L) gene product.
• In some embodiments, the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a umuD′, umuC, or recA730 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a dnaE486, dnaE1026, dnaX36, or dnaX2016 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutS538, mutS503, mutL705, mutL713, mutL(R261H), mutL4K307A), mutH(E56A), mutH(K79E), or mutH(K116E) gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR, mutH(E56A), mutL713, mutS503, mutSΔN, dinB, or polB gene product, or any combination thereof.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a ugi gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding an AID, AID (7), AID (7.3), AID (7.3.5). AID (7.3.3), AID (7.3.1), AID (7.3.2), APOBEC1, or CDA1 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutSΔN gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, yfjY, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A30IV)B, nrdEF, or nrdAB(P334L) gene product, or any combination thereof.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE74 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE486 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE1026 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX36 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX2016 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD12 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD14 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD16 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB(D156A) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a MAG1 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a AAG(Y127I-H136L) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Δ80-AAG(Y127I-H136L) gene product.
• In some embodiments, the expression construct is comprised in a plasmid. In some embodiments, the plasmid comprises a bacterial origin of replication. In some embodiments, the origin of replication is a cloDF13 origin of replication. In some embodiments, the plasmid comprises a nucleic acid sequence encoding a gene product conferring resistance to an antibiotic to a bacterial host cell. In some embodiments, the antibiotic is chloramphenicol, kanamycin, tetracycline, tetracycline, spectinomycin or apramycin, or ampicillin. Additional suitable origins of replication and antibiotics for use in embodiments of this disclosure will be apparent to those of skill in the art. The disclosure is not limited in this respect.
• In some embodiments, the expression construct comprises at least one inducible promoter controlling the expression of at least one nucleic acid sequence encoding the gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway, or any combination thereof. In some embodiments, the inducible prompter is an arabinose responsive promoter. In some embodiments, the arabinose responsive promoter is a P_BADpromoter. In some embodiments, the expression construct comprises a nucleic acid sequence encoding an arabinose operon regulatory protein. In some embodiments, the arabinose operon regulatory protein is araC. In some embodiments, the nucleic acid sequence encoding the arabinose operon regulatory protein is under the control of a weak promoter. In some embodiments, the weak promoter is a P_Cpromoter. Additional weak promoters will be apparent to those of skill in the art, and the present disclosure is not limited in this respect.
• In some embodiments, the expression construct comprises at least one codon-optimized nucleic acid sequence encoding a gene product. In some embodiments, the codon-optimized nucleic acid sequence comprises at least one codon of a naturally-occurring sequence encoding the gene product that has been replaced with a different codon encoding the same amino acid. In some embodiments, the at least one codon replacing the codon of the naturally occurring nucleic acid sequence corresponds to a tRNA expressed in a bacterial host cell. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a ribosome-binding site, and wherein at least one ribosome-binding site encoded by the expression construct is modified as compared to a naturally occurring ribosome binding site. In some embodiments, the modified ribosome binding site exhibits increased ribosome binding as compared to the naturally occurring ribosome binding site.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a gene product or a combination of such sequences listed in Table 2 orFIG. 7.
• Some aspects of this disclosure provide plasmids, cosmids, or artificial chromosomes (e.g., bacterial artificial chromosome, yeast artificial chromosomes, etc.) comprising an expression construct as disclosed herein.
• Some aspects of this disclosure provide a cell comprising an expression construct or a plasmid as provided herein. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell further comprises a selection plasmid or an accessory plasmid. In some embodiments, the cell is a host cell for a bacteriophage. In some embodiments, the cell is anE. colicell. In some embodiments, the cell is in a lagoon.
• Some aspects of this disclosure provide methods for modulating the mutation rate in a cell, e.g., in a host cell for a bacteriophage. In some embodiments, the method comprises contacting the cell with an expression construct or a plasmid as disclosed herein. In some embodiments, the expression construct comprises an inducible promoter, and the method further comprises culturing the host cell under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, and culturing the host cell under conditions suitable to induce expression from the inducible promoter comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose. In some embodiments, the method results in an at least 10-fold, at least 100-fold, at least 1000-fold, at least 10000-fold, at least 15000-fold, at least 200000-fold, at least 250000-fold, or at least 300000-fold increased mutation rate as compared to the mutation rate in the host cell in the absence of the expression construct.
• Some aspects of this disclosure provide methods for directed evolution using an expression construct provided herein. In some embodiments, the method comprises (a) contacting a population of host cells comprising a mutagenesis expression construct or plasmid as provided herein with a population of phage vectors comprising a gene to be evolved and deficient in at least one gene for the generation of infectious phage particles, wherein (1) the host cells are amenable to transfer of the vector; (2) the vector allows for expression of the gene to be evolved in the host cell, can be replicated by the host cell, and the replicated vector can transfer into a second host cell; (3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles of (a) in response to the activity of the gene to be evolved, and the level of gene product expression depends on the activity of the gene to be evolved; (b) incubating the population of host cells under conditions allowing for mutation of the gene to be evolved and the transfer of the vector comprising the gene to be evolved from host cell to host cell, wherein host cells are removed from the host cell population, and the population of host cells is replenished with fresh host cells that comprise the expression construct but do not harbor the vector, and (c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene to be evolved.
• In some embodiments, the expression construct comprises an inducible promoter, and the step of incubating (step (b)) comprises culturing the population of host cells under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, and the step of incubating (step (b)) comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose.
• In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a phage. In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an M13 phage.
• In some embodiments, the at least one gene for the generation of infectious phage particles comprises a sequence encoding a pIII protein. In some embodiments, the at least one gene for the generation of infectious phage particles comprises a full-length gIII gene. In some embodiments, the host cells comprise all phage genes except for the at least one gene for the generation of infectious phage particles in the form of a helper phage. In some embodiments, the phage genes comprised on the helper phage comprise pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, pX, and/or pXI.
• In some embodiments, the host cells comprise an accessory plasmid. In some embodiments, the accessory plasmid comprises an expression construct encoding the pIII protein under the control of a promoter that is activated by a gene product encoded by the gene to be evolved. In some embodiments, the host cells comprise the accessory plasmid, and together the helper phage and the accessory plasmid comprise all genes required for the generation of an infectious phage. In some embodiments, the method further comprises a negative selection for undesired activity of the gene to be evolved. In some embodiments, the host cells comprise an expression construct encoding a dominant-negative pII protein (pIII-neg). In some embodiments, expression of the pIII-neg protein is driven by a promoter the activity of which depends on an undesired function of the gene to be evolved.
• In some embodiments, step (b) comprises incubating the population of host cells for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive life cycles of the viral vector or phage. In some embodiments, the host cells areE. colicells.
• In some embodiments, the host cells are incubated in suspension culture. In some embodiments, the population of host cells is continuously replenished with fresh host cells that do not comprise the vector. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant number of cells in the cell population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector, viral, or phage load. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the cells in the cell population. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the frequency of host cells harboring the vector and/or of host cells not harboring the vector in the cell population. In some embodiments, the quantifying is by measuring the turbidity of the host cell culture, measuring the host cell density, measuring the wet weight of host cells per culture volume, or by measuring light extinction of the host cell culture.
• In some embodiments, the host cells are not exposed to a mutagen other than the mutagenesis expression construct or constructs provided herein. In some embodiments, the host cells are exposed to a mutagen. In some embodiments, the mutagen is ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), or t-butyl hydroperoxide (BHP) (CAS no. 75-91-2).
• In some embodiments, the method comprises a phase of diversifying the population of vectors by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the gene to be evolved in the absence of stringent selection for vectors having acquired a gain-of-function mutation in the gene to be evolved. In some embodiments, the method comprises a phase of stringent selection for a mutated replication product of the viral vector encoding an evolved gene.
• Some aspects of this disclosure provide kits comprising (a) a mutagenesis expression construct or plasmid as provided herein, wherein the expression construct comprises an inducible promoter controlling at least one of the nucleic acid sequences comprised in the expression construct; and (b) an inducing agent that induces expression from the inducible promoter. In some embodiments, the kit further comprises (c) a vector encoding an M13 phage backbone and a multiple cloning site for insertion of a nucleic acid sequence encoding a gene product to be evolved, wherein the vector or a replication product thereof can be packaged into infectious phage particles in the presence of other phage functions by suitable host cells, but lacks at least one gene required for the generation of infectious particles. In some embodiments, the kit further comprises (d) an accessory plasmid comprising a nucleic acid sequence encoding the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by a desired activity of the gene product to be evolved. In some embodiments, the kit further comprises (e) an accessory plasmid comprising a nucleic acid sequence encoding a dominant-negative version of the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by an undesired activity of the gene product to be evolved. In some embodiments, the kit further comprises a helper phage providing all phage functions except for the at least one gene required for the generation of infectious phage particles provided by the accessory plasmid of (d). In some embodiments, the helper phage or a replication product thereof cannot be packaged into infectious phage particles. In some embodiments, the kit comprises suitable host cells. In some embodiments, the host cells areE. colihost cells.
• The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIGS. 1A-IB. Mutagenesis plasmid (MP) design and effect on mutation rate in bacteria. The mutator genes tested are color-coded to indicate the canonical pathway being disrupted through the overexpression (FIG. 1A) or deletion (FIG. 1B) of that gene. All MPs express mutator genes from the arabinose-inducible P_BADpromoter, with each gene preceded by a ribosome-binding site to allow for translation from a single transcript. For (FIG. 1A), the mutagenesis rate μ_bp(substitutions per base pair of theE. coligenome per generation) was calculated using rifampin resistance under uninduced (25 mM glucose, white bars) and induced (25 mM glucose+25 mM arabinose, black bars). For (FIG. 1B), the rifampin resistance of XL1-Blue (white bar) and XL1-Red (black bar) was used to calculate μ_bp.
• FIGS. 2A-2C. Features of the MP system. (FIG. 2A) The dynamic range of MP6 was evaluated using increasing concentrations of arabinose (black circles) in the presence of 25 mM glucose in all cases. Treatment with 200 mM glucose only (dotted line) showed low mutagenesis under identical conditions. Comparison of the mutation rate under induced and uninduced conditions reveals a 35,000-fold dynamic range. Using the number of unique mutations found in rifampin-resistant rpoB alleles (21 sites), the average number of substitutions/genome/generation (μ_g) was calculated for (FIG. 2B) MG1655 ΔrecA::apra without an MP (white bar) vs. carrying MP1-6 under induced conditions (black bars), and (FIG. 2C) XL1-Blue (white bar) vs. XL1-Red (black bar).
• FIGS. 3A-3C. In vivo mutagenesis of M13 bacteriophage DNA. (FIG. 3A) S1030 cells (F⁺) carrying the indicated MPs were infected with M13 bacteriophage carrying a constitutive lacZ expression cassette (SP063), accompanied by the induction of the MP using arabinose, or suppression of the MP using glucose. (FIG. 3B) XL1-Blue or XL1-Red cells were transformed with purified SP063 DNA and recovered after overnight growth. (FIG. 3C) S1021 cells (identical to S1030, but lacking F′) carrying the indicated MPs were induced with arabinose or repressed with glucose for 2 h, transformed with purified SP063 DNA, and again induced with arabinose or repressed with glucose during recovery. For (FIG. 3A)-(FIG. 3C), progeny phage from the overnight propagation were plaqued on S1030 cells and stained using the X-Gal analog Bluo-Gal. The fraction of plaques that showed a white or light-blue lacZ⁻ phenotype reflecting loss-of-function mutation(s) is shown.
• FIGS. 4A-4I. Mutagenic spectra of the MPs. (FIGS. 4A-4D) The rpoB locus of single rifampin-resistant colonies was amplified by PCR and sequenced in both clusters I (aa 451-754) and II (aa 84-401). (FIG. 4E) Identities of the rifampin-resistant rpoB alleles from MP1, MP4, or MP6 mutagenesis, or using XL1-Red. The MPs yielded a wide distribution of mutation types, with the diversity of alleles strongly correlating with MP potency. (FIGS. 4F-4I) SP063 phage containing a constitutive lacZ expression cassette was propagated on the indicated mutator strain under induced conditions, and mutations were analyzed by high-throughput sequencing. In all cases, the number of sequenced mutations (n) is indicated for the MP and XL1-Red assays. * For (FIG. 4D) and (FIG. 4E), all sequenced XL1-Red rifampin-resistant colonies carried the identical F505S/S531F rpoB genotype.
• FIGS. 5A-5B. Comparison of MP6 and other mutagenesis approaches for the evolution of antibiotic resistance. (FIG. 5A) MG1655 ΔrecA::apra cells with or without MP6 were grown for 18-21 h under induced conditions. XL1-Blue and XL1-Red were grown for 18-21 h. (FIG. 5B) MG1655 ΔrecA::apra cells were treated using chemical mutagens as previously described 1321. In all cases, cultures were plated on the indicated antibiotics following overnight culture in the absence of any selection. The numbers in parenthesis indicate the antibiotic concentrations used in μg/mL. The fraction of cells resistant to each antibiotic was calculated relative to the total number of cells on plates without antibiotics. Resistance to norfloxacin was not detected for any of the tested strains or treatments. XL1-Blue and XL1-Red are both inherently resistant to tetracycline and metronidazole, so no comparison is shown for either antibiotic. See Table 6 for full antibiotic resistance data.
• FIGS. 6A-6B. Continuous evolution of P_T3-active RNAP variants. S1030 cells carrying an accessory plasmid (AP) encoding P_T3upstream of M13 bacteriophage geneIII and either MP1a or MP6a were infected with selection phage (SP) carrying wild-type T7 RNAP under conditions in which selection stringency was high (0 ng/mL ATc) or moderate (30 ng/mL ATc). (FIG. 6A) Total phage titers at 10 h and 20 h after lagoon inoculation with the SP encoding T7 RNAP. (FIG. 6B) Titers of phage encoding evolved RNAP variants active on P_T3. The limit of detection is 50 pfu/mL.
• FIG. 7. Summary of major pathways that influenceE. coliDNA replication fidelity. Steps during replication and mutation correction are grouped according to their mechanisms of action. Methylated DNA is shown in black, unmethylated DNA is shown in grey, and the mutation to be corrected is depicted as “M”. Gene superscripts denote if a mutator phenotype results upon gene deletion [43] (1), gene overexpression [17, 20, 25, 46, 48] (2) or modification of the chromosomal allele to circumvent potential knockout lethality [17, 20, 25, 46, 48, 49] (3).
• FIG. 8. Cryptic σ⁷⁰promoter at the 3′ end of the dnaQ926 ORF. Annotated sequence of the predicted σ⁷⁰promoter in MP3 bridging the 3′ end of the dnaQ926 ORF and RBS driving the dam ORF. Sequences correspond, from top to bottom, to SEQ ID NOs: 12-15.
• FIG. 9. Cryptic σ⁷⁰promoters at the 3′ end of the seqA ORF. Annotated sequence of the predicted σ⁷⁰promoters in MP5 bridging the 3′ end of the seqA ORF and native RBS driving the ugi ORF. Sequences correspond, from top to bottom, to SEQ ID NOs: 16-19.
• FIGS. 10A-10B. Effect of MPs on host viability under induced conditions. (FIG. 10A) Relative cell viability was calculated as the fractional cell titer following arabinose induction as compared to the uninduced control for each MP. Viability is anti-correlated with mutagenic potency at high levels. (FIG. 10B) The XL1-Red strain shows the expected level of viability (compared to the control, XL1-Blue) given its mutagenic potency as compared to the designed MPs.
• FIG. 11. Relationship between host viability and induced levels of mutagenesis for all MPs. Low potency MPs which induce up to ˜1×10⁻⁶, substitutions/bp/generation were well tolerated by theE. coliMG1655 ΔrecA::apra host, while higher levels of mutagenesis generally resulted in a reduced host viability, as expected. This inflection point corresponds to ˜4.6 substitutions/genome/generation for wild-typeE. coliMG/566 (genome size=4.64×10⁶bp).
• FIG. 12. Relationship between uninduced and induced levels of mutagenesis for all MPs. Higher levels of background (uninduced mutagenesis) were generally accompanied by an increase in overall MP mutagenesis upon induction. The full data set is provided in Table 2.
• FIG. 13. Plaque assay of the lacZ-carrying M13 phage SP063. SP063 carries the wild-typeE. coliβ-galactosidase gene with a consensus ribosome-binding site directly downstream of geneIII. Plating using soft agar containing S1030 cells in the presence of the X-Gal analog Bluo-Gal (Life Technologies) results in a strong, deep blue plaques (shown here as dark circles).
• FIGS. 14A-14B. Optimization of phage inoculant for optimal expansion and mutagenesis. (FIG. 14A)E. coliS1030 carrying MP6 were induced with arabinose during log-phase growth and concomitantly infected with serially diluted SP063 phage. Phage were titered after overnight propagation and the fold expansion of the phage population was determined. (FIG. 14B) The percentage of white and light blue (lacZ⁻) plaques in the presence of Bluo-Gal suggests a correlation between phage population expansion and mutagenesis efficiency.
• FIGS. 15A-15B. Effect of MP pre-induction on phage mutagenesis. (FIG. 15A)E. coliS1030 carrying MP6 were induced with arabinose during log-phase growth and concomitantly infected with SP063 phage at defined titers. Alternatively, infection was delayed for 1 h or 2 h. (FIG. 15B) Phage were titered after overnight propagation and the percentage of white plaques in the presence of Bluo-Gal was counted.
• FIG. 16. Analysis of F′ episomal mutations rates using various MPs. The frequency of lacZ+ revertants is the fraction of colonies surviving on lactose as the sole carbon source as compared to the total colony count (colonies that survive on glucose as sole carbon source). Each strain reports the MP's ability to increase the frequency of a specific mutation type.
• FIGS. 17A-17D. Mutagenic spectra of commonly used mutagenesis techniques. Previously reported mutagenic spectra of four commonly used mutagenesis methods are shown:FIG. 17A: LF Pol I⁹;FIG. 17B: mutA³⁷;FIG. 17C: Mutazyme II³⁸;FIG. 17D: and EMS³⁹.
• FIG. 18. Activity of T7 RNAP on cognate and non-cognate promoters. Log-phase S1030 cells carrying accessory plasmids (APs) with a geneIII cassette with an upstream phage shock protein (PSP). T7, hybrid T7/T3, or T3 promoter were infected with selection phage (SPs) carrying the wild-type T7 RNAP. The fraction of output phage vs. input phage indirectly reports on the activity of the T7 RNAP on the various promoters. Enrichment factors of ˜100 or less indicate extremely weak to non-existent activity.
• FIG. 19. Single-phage plaque sequencing of P_T3-active SPs. Single phage plaques at 10 h of PACE using the P_T3AP were isolated and subjected to Sanger sequencing. All clones carried T7 RNAP variants with conserved mutations known to confer activity on P_T3(blue), as well as additional mutations that may further enhance activity (black). Silent mutations were also detected (red).
• FIG. 20. DP1 Vector Map. A schematic depiction of one embodiment of a DP1 drift plasmid is provided, referenced herein as SEQ ID NO: 27. This embodiment comprises araC, dnaQ926, umuD′, umuC, recA730, and an anhydrotetracycline (ATc)-dependent drift promoter.
• FIG. 21. DP2 Vector Map. A schematic depiction of one embodiment of a DP2 drift vector is provided, referenced herein as SEQ ID NO: 28. This embodiment comprises araC, dnaQ926, and an anhydrotetracycline (ATc)-dependent drift promoter.
• FIG. 22. DP3 Vector Map. A schematic depiction of one embodiment of a DP3 drift vector is provided, referenced herein as SEQ ID NO: 29. This embodiment comprises araC, dnaQ926, dam, and an anhydrotetracycline (ATc)-dependent drift promoter.
• FIG. 23. DP4 Vector Map. A schematic depiction of one embodiment of a DP4 drift vector is provided, referenced herein as SEQ ID NO: 33. This embodiment comprises araC, dnaQ926, dam, seqA, and an anhydrotetracycline (ATc)-dependent drift promoter.
• FIG. 24. DP5 Vector Map. A schematic depiction of one embodiment of a DP5 drift vector is provided, referenced herein as SEQ ID NO: 34. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and/or pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter.
• FIG. 25. DP6 Vector Map. A schematic depiction of one embodiment of a DP6 drift vector is provided, referenced herein as SEQ ID NO: 35. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and/or pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter.
• FIG. 26. MP1 Vector Map. A schematic depiction of one embodiment of a MP1 mutagenesis vector is provided, referenced herein as SEQ ID NO: 43. This embodiment comprises dnaQ926, umuD′, umuC, and recA730.
• FIG. 27. MP2 Vector Map. A schematic depiction of one embodiment of a MP2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 44. This embodiment comprises dnaQ926.
• FIG. 28. MP3 Vector Map. A schematic depiction of one embodiment of a MP3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 45. This embodiment comprises araC, dnaQ926, and dam.
• FIG. 29. MP4 Vector Map. A schematic depiction of one embodiment of a MP4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 46. This embodiment comprises araC, dnaQ926, dam, and seqA.
• FIG. 30. MP5 Vector Map. A schematic depiction of one embodiment of a MP5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 47. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1
• FIG. 31. MP6 Vector Map. A schematic depiction of one embodiment of a MP6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 48. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 32. MP-B2 Vector Map. A schematic depiction of one embodiment of a MP-B2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 49. This embodiment comprises dnaE374.
• FIG. 33. MP-B4 Vector Map. A schematic depiction of one embodiment of a MP-B4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 50. This embodiment comprises dnaE486.
• FIG. 34. MP-B5 Vector Map. A schematic depiction of one embodiment of a MP-B5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 51. This embodiment comprises araC, and dnaE1026.
• FIG. 35. MP-C2 Vector Map. A schematic depiction of one embodiment of a MP-C2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 52. This embodiment comprises araC, and dnaX36.
• FIG. 36. MP-C3 Vector Map. A schematic depiction of one embodiment of a MP-C3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 53. This embodiment comprises araC, and dnaX2016.
• FIG. 37. MP-D3 Vector Map. A schematic depiction of one embodiment of a MP-D3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 54. This embodiment comprises araC, and dnaE486.
• FIG. 38. MP-D4 Vector Map. A schematic depiction of one embodiment of a MP-D4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 55. This embodiment comprises araC, dnaQ926, and dnaE1026.
• FIG. 39. MP-E Vector Map. A schematic depiction of one embodiment of a MP-E mutagenesis vector is provided, referenced herein as SEQ ID NO: 56. This embodiment comprises araC, dnaQ926, and dnaX36.
• FIG. 40. MP-E2 Vector Map. A schematic depiction of one embodiment of a MP-E2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 57. This embodiment comprises araC, dnaQ926, and dnaX2016.
• FIG. 41. MP-F2 Vector Map. A schematic depiction of one embodiment of a MP-F2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 58. This embodiment comprises araC, dnaQ926, dnaX2016, and mutS538′.
• FIG. 42. MP-F3 Vector Map. A schematic depiction of one embodiment of a MP-F3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 59. This embodiment comprises araC, dnaQ926, and mutS503′.
• FIG. 43. MP-H Vector Map. A schematic depiction of one embodiment of a MP-H mutagenesis vector is provided, referenced herein as SEQ ID NO: 60. This embodiment comprises araC, dnaQ926, and mutL705.
• FIG. 44. MP-H2 Vector Map. A schematic depiction of one embodiment of a MP-H2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 61. This embodiment comprises araC, dnaQ926, and mutL713.
• FIG. 45. MP-H3 Vector Map. A schematic depiction of one embodiment of a MP-H3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 62. This embodiment comprises araC, dnaQ926, and mutL(R261H).
• FIG. 46. MP-H4 Vector Map. A schematic depiction of one embodiment of a MP-H4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 63. This embodiment comprises araC, dnaQ926, and mutL(K307A).
• FIG. 47. MP-I Vector Map. A schematic depiction of one embodiment of a MP-I mutagenesis vector is provided, referenced herein as SEQ ID NO: 64. This embodiment comprises araC, dnaQ926, and mutH (E564).
• FIG. 48. MP-I2 Vector Map. A schematic depiction of one embodiment of a MP-I2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 65. This embodiment comprises araC, dnaQ926, and mutH (K79E).
• FIG. 49. MP-I3 Vector Map. A schematic depiction of one embodiment of a MP-I3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 66. This embodiment comprises araC, dnaQ926, and mutH (K116E).
• FIG. 50. MP-J Vector Map. A schematic depiction of one embodiment of a MP-J mutagenesis vector is provided, referenced herein as SEQ ID NO: 67. This embodiment comprises araC, and rpsD12.
• FIG. 51. MP-J2 Vector Map. A schematic depiction of one embodiment of a MP-J2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 68. This embodiment comprises araC, and rpsD14.
• FIG. 52. MP-J3 Vector Map. A schematic depiction of one embodiment of a MP-J3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 69. This embodiment comprises araC, and rpsD16.
• FIG. 53. MP-K10 Vector Map. A schematic depiction of one embodiment of a MP-K10 mutagenesis vector is provided, referenced herein as SEQ ID NO: 70. This embodiment comprises araC, dnaQ926, dam, seqA, and emrR.
• FIG. 54. MP-K11 Vector Map. A schematic depiction of one embodiment of a MP-K11 mutagenesis vector is provided, referenced herein as SEQ ID NO: 71. This embodiment comprises araC, dnaQ926, dam, seqA, and mutSdeIN (del 2-98).
• FIG. 55. MP-K12 Vector Map. A schematic depiction of one embodiment of a MP-K12 mutagenesis vector is provided, referenced herein as SEQ ID NO: 72. This embodiment comprises araC, dnaQ926, dam, seqA, and dinB.
• FIG. 56. MP-K13 Vector Map. A schematic depiction of one embodiment of a MP-K13 mutagenesis vector is provided, referenced herein as SEQ ID NO: 73. This embodiment comprises araC, dnaQ926, dam, seqA, and polB.
• FIG. 57. MP-K14 Vector Map. A schematic depiction of one embodiment of a MP-K14 mutagenesis vector is provided, referenced herein as SEQ ID NO: 74. This embodiment comprises araC, dnaQ926, dam, and seqA (RBS+).
• FIG. 58. MP-K7 Vector Map. A schematic depiction of one embodiment of a MP-K7 mutagenesis vector is provided, referenced herein as SEQ ID NO: 75. This embodiment comprises araC, dnaQ926, dam, and emrR.
• FIG. 59. MP-K9 Vector Map. A schematic depiction of one embodiment of a MP-K9 mutagenesis vector is provided, referenced herein as SEQ ID NO: 76. This embodiment comprises araC, dnaQ926, dam, and mutSdeIN(2-98).
• FIG. 60. MP-L Vector Map. A schematic depiction of one embodiment of a MP-L mutagenesis vector is provided, referenced herein as SEQ ID NO: 77. This embodiment comprises araC, and polB.
• FIG. 61. MP-L2 Vector Map. A schematic depiction of one embodiment of a MP-L2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 78. This embodiment comprises araC, and polB(D156A).
• FIG. 62. MP-P Vector Map. A schematic depiction of one embodiment of a MP-P mutagenesis vector is provided, referenced herein as SEQ ID NO: 79. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutH(E56A).
• FIG. 63. MP-P11 Vector Map. A schematic depiction of one embodiment of a MP-P11 mutagenesis vector is provided, referenced herein as SEQ ID NO: 80. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1, mutSdeIN(del-29).
• FIG. 64. MP-P3 Vector Map. A schematic depiction of one embodiment of a MP-P3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 81. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutL713.
• FIG. 65. MP-P4 Vector Map. A schematic depiction of one embodiment of a MP-P4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 82. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutS503′.
• FIG. 66. MP-P5 Vector Map. A schematic depiction of one embodiment of a MP-P5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 83. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and mutSdeIN(del 2-98).
• FIG. 67. MP-P6 Vector Map. A schematic depiction of one embodiment of a MP-P6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 84. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and dinB.
• FIG. 68. MP-P7 Vector Map. A schematic depiction of one embodiment of a MP-P7 mutagenesis vector is provided, referenced herein as SEQ ID NO: 85. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, and polB.
• FIG. 69. MP-P8 Vector Map. A schematic depiction of one embodiment of a MP-P8 mutagenesis vector is provided, referenced herein as SEQ ID NO: 86. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID.
• FIG. 70. MP-P9 Vector Map. A schematic depiction of one embodiment of a MP-P9 mutagenesis vector is provided, referenced herein as SEQ ID NO: 87. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and mAPOBEC1.
• FIG. 71. MP-Q Vector Map. A schematic depiction of one embodiment of a MP-Q mutagenesis vector is provided, referenced herein as SEQ ID NO: 88. This embodiment comprises araC, dnaQ926, dam, seqA, and rsmE.
• FIG. 72. MP-Q10 Vector Map. A schematic depiction of one embodiment of a MP-Q10 mutagenesis vector is provided, referenced herein as SEQ ID NO: 89. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdA(A65V)B.
• FIG. 73. MP-Q11 Vector Map. A schematic depiction of one embodiment of a MP-Q11 mutagenesis vector is provided, referenced herein as SEQ ID NO: 90. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdA(A301V)B.
• FIG. 74. MP-Q12 Vector Map. A schematic depiction of one embodiment of a MP-Q12 mutagenesis vector is provided, referenced herein as SEQ ID NO: 91. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdAB(P334L).
• FIG. 75. MP-Q13 Vector Map. A schematic depiction of one embodiment of a MP-Q13 mutagenesis vector is provided, referenced herein as SEQ ID NO: 92. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdEF.
• FIG. 76. MP-Q2 Vector Map. A schematic depiction of one embodiment of a MP-Q2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 93. This embodiment comprises araC, dnaQ926, dam, seqA, and cchA.
• FIG. 77. MP-Q3 Vector Map. A schematic depiction of one embodiment of a MP-Q3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 94. This embodiment comprises araC, dnaQ926, dam, seqA, and yffI.
• FIG. 78. MP-Q4 Vector Map. A schematic depiction of one embodiment of a MP-Q4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 95. This embodiment comprises araC, dnaQ926, dam, seqA, and yfjY.
• FIG. 79. MP-Q5 Vector Map. A schematic depiction of one embodiment of a MP-Q5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 96. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and hsAID.
• FIG. 80. MP-Q6 Vector Map. A schematic depiction of one embodiment of a MP-Q6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 97. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and mAPOBEC1.
• FIG. 81. MP-Q8 Vector Map. A schematic depiction of one embodiment of a MP-Q8 mutagenesis vector is provided, referenced herein as SEQ ID NO: 98. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdAB.
• FIG. 82. MP-Q9 Vector Map. A schematic depiction of one embodiment of a MP-Q9 mutagenesis vector is provided, referenced herein as SEQ ID NO: 99. This embodiment comprises araC, dnaQ926, dam, seqA, and nrdA(H59A)B.
• FIG. 83. MP-R Vector Map. A schematic depiction of one embodiment of a MP-R mutagenesis vector is provided, referenced herein as SEQ ID NO: 100. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and hsAID.
• FIG. 84. MP-R2 Vector Map. A schematic depiction of one embodiment of a MP-R2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 101. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and mAPOBEC1.
• FIG. 85. MP-R3 Vector Map. A schematic depiction of one embodiment of a MP-R3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 102. This embodiment comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1.
• FIG. 86. MP-R4 Vector Map. A schematic depiction of one embodiment of a MP-R4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 103. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID.
• FIG. 87. MP-R5 Vector Map. A schematic depiction of one embodiment of a MP-R5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 104. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and mAPOBEC1.
• FIG. 88. MP-R6 Vector Map. A schematic depiction of one embodiment of a MP-R6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 105. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 89. MP-S Vector Map. A schematic depiction of one embodiment of a MP-S mutagenesis vector is provided, referenced herein as SEQ ID NO: 106. This embodiment comprises araC, dnaQ926, and MAG1.
• FIG. 90. MP-S2 Vector Map. A schematic depiction of one embodiment of a MP-S2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 107. This embodiment comprises araC, dnaQ926, and AAG(Y127I-H136L).
• FIG. 91. MP-S3 Vector Map. A schematic depiction of one embodiment of a MP-S3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 108. This embodiment comprises araC, dnaQ926, and del80-AAG(Y127I-H136L).
• FIG. 92. MP-T Vector Map. A schematic depiction of one embodiment of a MP-T mutagenesis vector is provided, referenced herein as SEQ ID NO: 109. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G).
• FIG. 93. MP-T2 Vector Map. A schematic depiction of one embodiment of a MP-T2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 110. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).
• FIG. 94. MP-T3 Vector Map. A schematic depiction of one embodiment of a MP-T3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 111. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).
• FIG. 95. MP-T4 Vector Map. A schematic depiction of one embodiment of a MP-T4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 112. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).
• FIG. 96. MP-T5 Vector Map. A schematic depiction of one embodiment of a MP-T5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 113. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).
• FIG. 97. MP-T6 Vector Map. A schematic depiction of one embodiment of a MP-T6 mutagenesis vector is provided, referenced herein as SEQ ID NO: 114. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and hsAID (K10E/E156G/T821).
• FIG. 98. MP-U Vector Map. A schematic depiction of one embodiment of a MP-U mutagenesis vector is provided, referenced herein as SEQ ID NO: 115. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 99. MP-U2 Vector Map. A schematic depiction of one embodiment of a MP-U2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 116. This embodiment comprises araC, dnaQ926 RBS+, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 100. MP-U3 Vector Map. A schematic depiction of one embodiment of a MP-U3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 117. This embodiment comprises araC, dnaQ926, dam, seqA RBS+, emrR, ugi and pmCDA1.
• FIG. 101. MP-U4 Vector Map. A schematic depiction of one embodiment of a MP-U4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 118. This embodiment comprises araC, dnaQ926, dam, seqA, emrR, ugi RBS+ and pmCDA1.
• FIG. 102. MP-V Vector Map. A schematic depiction of one embodiment of a MP-V mutagenesis vector is provided, referenced herein as SEQ ID NO: 119. This embodiment comprises araC, dnaQ-BRM1, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 103. MP-V2 Vector Map. A schematic depiction of one embodiment of a MP-V2 mutagenesis vector is provided, referenced herein as SEQ ID NO: 120. This embodiment comprises araC, dnaQ-BRM1, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 104. MP-V3 Vector Map. A schematic depiction of one embodiment of a MP-V3 mutagenesis vector is provided, referenced herein as SEQ ID NO: 121. This embodiment comprises araC, dnaQ-BR11, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 105. MP-V4 Vector Map. A schematic depiction of one embodiment of a MP-V4 mutagenesis vector is provided, referenced herein as SEQ ID NO: 122. This embodiment comprises araC, dnaQ-BR6, dam, seqA, emrR, ugi and pmCDA1.
• FIG. 106. MP-V5 Vector Map. A schematic depiction of one embodiment of a MP-V5 mutagenesis vector is provided, referenced herein as SEQ ID NO: 123. This embodiment comprises araC, dnaQ-BR1, dam, seqA, emrR, ugi and pmCDA1.
DEFINITIONS
• The term “accessory plasmid,” as used herein, refers to a plasmid comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter. In the context of continuous evolution of genes, transcription from the conditional promoter of the accessory plasmid is typically activated, directly or indirectly, by a function of the gene to be evolved. Accordingly, the accessory plasmid serves the function of conveying a competitive advantage to those viral vectors in a given population of viral vectors that carry a version of the gene to be evolved able to activate the conditional promoter or able to activate the conditional promoter more strongly than other versions of the gene to be evolved. In some embodiments, only viral vectors carrying an “activating” version of the gene to be evolved will be able to induce expression of the gene required to generate infectious viral particles in the host cell, and, thus, allow for packaging and propagation of the viral genome in the flow of host cells. Vectors carrying non-activating versions of the gene to be evolved, on the other hand, will not induce expression of the gene required to generate infectious viral vectors, and, thus, will not be packaged into viral particles that can infect fresh host cells.
• The term “cellstat,” as used herein, refers to a culture vessel comprising host cells, in which the number of cells is substantially constant over time.
• The term “continuous evolution,” as used herein, refers to an evolution process, in which a population of nucleic acids encoding a gene to be evolved is subjected to multiple rounds of (a) replication, (b) mutation, and (c) selection to produce a desired evolved version of the gene to be evolved that is different from the original version of the gene, for example, in that a gene product, such as, e.g., an RNA or protein encoded by the gene, exhibits a new activity not present in the original version of the gene product, or in that an activity of a gene product encoded by the original gene to be evolved is modulated (increased or decreased). The multiple rounds can be performed without investigator intervention, and the steps (a)-(c) can be carried out simultaneously. Typically, the evolution procedure is carried out in vitro, for example, using cells in culture as host cells. In general, a continuous evolution process provided herein relies on a system in which a gene encoding a gene product of interest is provided in a nucleic acid vector that undergoes a life-cycle including replication in a host cell and transfer to another host cell, wherein a critical component of the life-cycle is deactivated and reactivation of the component is dependent upon an activity of the gene to be evolved that is a result of a mutation in the nucleic acid vector.
• The term “flow”, as used herein in the context of host cells, refers to a stream of host cells, wherein fresh host cells not harboring the transfer vector (e.g., the viral vector encoding the gene to be evolved) are being introduced into a host cell population, for example, a host cell population in a lagoon, remain within the population for a limited time, and are then removed from the host cell population. In a simple form, a host cell flow may be a flow through a tube, or a channel, for example, at a controlled rate. In other embodiments, a flow of host cells is directed through a lagoon that holds a volume of cell culture media and comprises an inflow and an outflow. The introduction of fresh host cells may be continuous or intermittent and removal may be passive, e.g., by overflow, or active, e.g., by active siphoning or pumping. Removal further may be random, for example, if a stirred suspension culture of host cells is provided, removed liquid culture media will contain freshly introduced host cells as well as cells that have been a member of the host cell population within the lagoon for some time. Even though, in theory, a cell could escape removal from the lagoon indefinitely, the average host cell will remain only for a limited period of time within the lagoon, which is determined mainly by the flow rate of the culture media (and suspended cells) through the lagoon. Since the viral vectors replicate in a flow of host cells, in which fresh, uninfected host cells are provided while infected cells are removed, multiple consecutive viral life cycles can occur without investigator interaction, which allows for the accumulation of multiple advantageous mutations in a single evolution experiment.
• The term “fresh,” as used herein in the context of host cells, and used interchangeably with the terms “non-infected” or “uninfected” in the context of host cells of viral vectors, refers to a host cell that does not harbor the vector or, in the context of viral vectors, has not been infected by the viral vector comprising a gene to be evolved as used in a continuous evolution process provided herein. A fresh host cell can, however, have been infected by a viral vector unrelated to the vector to be evolved or by a vector of the same or a similar type but not carrying the gene of interest.
• The term “gene of interest” or “gene to be evolved,” as used herein, refers to a nucleic acid construct comprising a nucleotide sequence encoding a gene product, e.g., an RNA or a protein, to be evolved in a continuous evolution process as provided herein. The term includes any variations of a gene of interest that are the result of a continuous evolution process according to methods provided herein. For example, in some embodiments, a gene of interest is a nucleic acid construct comprising a nucleotide sequence encoding an RNA or protein to be evolved, cloned into a viral vector, for example, a phage genome, so that the expression of the encoding sequence is under the control of one or more promoters in the viral genome. In other embodiments, a gene of interest is a nucleic acid construct comprising a nucleotide sequence encoding an RNA or protein to be evolved and a promoter operably linked to the encoding sequence. When cloned into a viral vector, for example, a phage genome, the expression of the encoding sequence of such genes of interest is under the control of the heterologous promoter and, in some embodiments, may also be influenced by one or more promoters comprised in the viral genome. In some embodiments, the term “gene of interest” or “gene to be evolved refers to a nucleic acid sequence encoding a gene product to be evolved, without any additional sequences. In some embodiments, the term also embraces additional sequences associated with the encoding sequence, such as, for example, intron, promoter, enhancer, or polyadenylation signal sequences.
• The term “helper phage,” as used herein interchangeable with the terms “helper phagemid” and “helper plasmid,” refers to a nucleic acid construct comprising a phage gene required for the phage life cycle, or a plurality of such genes, but lacking a structural element required for genome packaging into a phage particle. For example, a helper phage may provide a wild-type phage genome lacking a phage origin of replication. In some embodiments, a helper phage is provided that comprises a gene required for the generation of phage particles, but lacks a gene required for the generation of infectious particles, for example, a full-length pIII gene. In some embodiments, the helper phage provides only some, but not all, genes for the generation of infectious phage particles. Helper phages are useful to allow modified phages that lack a gene for the generation of infectious phage particles to complete the phage life cycle in a host cell. Typically, a helper phage will comprise the genes for the generation of infectious phage particles that are lacking in the phage genome, thus complementing the phage genome. In the continuous evolution context, the helper phage typically complements the selection phage, but both lack a phage gene required for the production of infectious phage particles.
• The terms “high copy number plasmid” and “low copy number plasmid” are art-recognized, and those of skill in the art will be able to ascertain whether a given plasmid is a high or low copy number plasmid. In some embodiments, a low copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 5 to about 100. In some embodiments, a very low copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 1 to about 10. In some embodiments, a very low copy number accessory plasmid is a single-copy per cell plasmid. In some embodiments, a high copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 100 to about 5000.
• The term “host cell,” as used herein, refers to a cell that can host, replicate, and transfer a phage vector useful for a continuous evolution process as provided herein. In embodiments where the vector is a viral vector, a suitable host cell is a cell that can be infected by the viral vector, can replicate it, and can package it into viral particles that can infect fresh host cells. A cell can host a viral vector if it supports expression of genes of viral vector, replication of the viral genome, and/or the generation of viral particles. One criterion to determine whether a cell is a suitable host cell for a given viral vector is to determine whether the cell can support the viral life cycle of a wild-type viral genome that the viral vector is derived from. For example, if the viral vector is a modified M13 phage genome, as provided in some embodiments described herein, then a suitable host cell would be any cell that can support the wild-type M13 phage life cycle. Suitable host cells for viral vectors useful in continuous evolution processes are well known to those of skill in the art, and the disclosure is not limited in this respect.
• The term “infectious viral particle,” as used herein, refers to a viral particle able to transport the viral genome it comprises into a suitable host cell. Not all viral particles are able to transfer the viral genome to a suitable host cell. Particles unable to accomplish this are referred to as non-infectious viral particles. In some embodiments, a viral particle comprises a plurality of different coat proteins, wherein one or some of the coat proteins can be omitted without compromising the structure of the viral particle. In some embodiments, a viral particle is provided in which at least one coat protein cannot be omitted without the loss of infectivity. If a viral particle lacks a protein that confers infectivity, the viral particle is not infectious. For example, an M13 phage particle that comprises a phage genome packaged in a coat of phage proteins (e.g., pVIII) but lacks pIII (protein III) is a non-infectious M13 phage particle because pIII is essential for the infectious properties of M13 phage particles.
• The term “lagoon.” as used herein, refers to a culture vessel or bioreactor through which a flow of host cells is directed. When used for a continuous evolution process as provided herein, a lagoon typically holds a population of host cells and a population of viral vectors replicating within the host cell population, wherein the lagoon comprises an outflow through which host cells are removed from the lagoon and an inflow through which fresh host cells are introduced into the lagoon, thus replenishing the host cell population.
• The term “mutagen,” as used herein, refers to an agent that induces mutations or increases the rate of mutation in a given biological system, for example, a host cell, to a level above the naturally occurring level of mutation in that system. Some exemplary mutagens useful for continuous evolution procedures are provided elsewhere herein, and other useful mutagens will be evident to those of skill in the art. Useful mutagens include, but are not limited to, ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), and t-butyl hydroperoxide (BHP) (CAS no. 75-91-2). Additional mutagens can be used in continuous evolution procedures as provided herein, and the invention is not limited in this respect.
• The term “mutagenesis plasmid,” as used herein, refers to a plasmid comprising a nucleic acid sequence encoding a gene product or a combination of gene products that act(s) as a mutagen.
• The term “nucleic acid,” as used herein, refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).
• The term “phage,” as used herein interchangeably with the term “bacteriophage,” refers to a virus that infects bacterial cells. Typically, phages consist of an outer protein capsid enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA, in either linear or circular form. Phages and phage vectors are well known to those of skill in the art and non-limiting examples of phages that are useful for carrying out the methods provided herein are λ (Lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, Phi X174, N4, Φ6, and Φ29. In certain embodiments, the phage utilized in the present invention is M13. Additional suitable phages and host cells will be apparent to those of skill in the art and the invention is not limited in this aspect. For an exemplary description of additional suitable phages and host cells, see Elizabeth Kutter and Alexander Sulakvelidze:Bacteriophages: Biology and Applications. CRC Press; 1^stedition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski:Bacteriophages; Methods and Protocols, Volume1: Isolation, Characterization, and Interactions(Methods in Molecular Biology) Humana Press; 1^stedition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski:Bacteriophages: Methods and Protocols, Volume2: Molecular and Applied Aspects(Methods in Molecular Biology) Humana Press; 1^stedition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable phages and host cells as well as methods and protocols for isolation, culture, and manipulation of such phages).
• The term “phage-assisted continuous evolution (PACE),” as used herein, refers to continuous evolution that employs phage as viral vectors. PACE technology has been described previously, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, each of which is incorporated herein by reference.
• The term “promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule “inducer” for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect.
• The term “protein,” as used herein refers to a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptide of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, cco.caltech.edu/˜dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be just a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these.
• The term “replication product,” as used herein, refers to a nucleic acid that is the result of viral genome replication by a host cell. This includes any viral genomes synthesized by the host cell from a viral genome inserted into the host cell. The term includes non-mutated as well as mutated replication products.
• The term “selection phage,” as used herein interchangeably with the term “selection plasmid,” refers to a modified phage that comprises a gene of interest to be evolved and lacks a full-length gene encoding a protein required for the generation of infectious phage particles. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a phage gene encoding a protein required for the generation of infectious phage particles, e.g., gI, gII, gIII, gIV, gV, gVI, gVII, gVIII, gIX, or gX, or any combination thereof. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a gene encoding a protein required for the generation of infective phage particles, e.g., the gIII gene encoding the pIII protein.
• The terms “small molecule” and “organic compound” are used interchangeably herein and refer to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, an organic compound contains carbon. An organic compound may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, or heterocyclic rings). In some embodiments, organic compounds are monomeric and have a molecular weight of less than about 1500 g/mol. In certain embodiments, the molecular weight of the small molecule is less than about 1000 g/mol or less than about 500 g/mol. In certain embodiments, the small molecule is a therapeutic drug or drug candidate, for example, a drug or drug candidate that is in clinical or pre-clinical trials or that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.
• The term “turbidostat,” as used herein, refers to a culture vessel comprising host cells in suspension culture, in which the turbidity of the culture medium is substantially essentially constant over time. In some embodiments, the turbidity of a suspension culture, for example, of bacterial cells, is a measure for the cell density in the culture medium. In some embodiments, a turbidostat comprises an inflow of fresh media and an outflow, and a controller that regulates the flow into and/or out of the turbidostat based on the turbidity of the suspension culture in the turbidostat.
• The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.
• The term “viral life cycle,” as used herein, refers to the viral reproduction cycle comprising insertion of the viral genome into a host cell, replication of the viral genome in the host cell, and packaging of a replication product of the viral genome into a viral particle by the host cell.
• The term “viral particle,” as used herein, refers to a viral genome, for example, a DNA or RNA genome, that is associated with a coat of a viral protein or proteins, and, in some cases, with an envelope of lipids. For example, a phage particle comprises a phage genome packaged into a protein encoded by the wild type phage genome.
• The term “viral vector.” as used herein, refers to a nucleic acid comprising a viral genome that, when introduced into a suitable host cell, can be replicated and packaged into viral particles able to transfer the viral genome into another host cell. The term viral vector extends to vectors comprising truncated or partial viral genomes. For example, in some embodiments, a viral vector is provided that lacks a gene encoding a protein essential for the generation of infectious viral particles. In suitable host cells, for example, host cells comprising the lacking gene under the control of a conditional promoter, however, such truncated viral vectors can replicate and generate viral particles able to transfer the truncated viral genome into another host cell. In some embodiments, the viral vector is a phage, for example, a filamentous phage (e.g., an M13 phage). In some embodiments, a viral vector, for example, a phage vector, is provided that comprises a gene of interest to be evolved.
DETAILED DESCRIPTIONIntroduction
• Some aspects of this disclosure provide systems, vectors, and methods for modulating the mutation rates in cells, for example, in bacterial host cells. In some embodiments, the present disclosure provides mutagenesis vectors, sometimes referred to herein as mutagenesis expression constructs or, if in the form of a plasmid, as mutagenesis plasmids, that mediate highly potent, broad-spectrum, and controllable mutagenesis in bacterial cells. These vectors can be used in many bacterial strains, including, but not limited to, mostE. colistrains, to mutagenize chromosomal, episomal, or viral DNA, enabling high-efficiency mutation, for example, during directed evolution, obviating the need to create random DNA libraries in vitro, and bypassing transformation efficiency bottlenecks. Some advantages of the systems, vectors, and methods for modulating the mutation rates in bacteria as provided herein include, for example, the ability to evolve a gene of interest in a significantly reduced time frame as compared to other mutagenic technologies, and the fact that the mutagenesis-inducing vectors and constructs are not harmful to humans, in contrast to many other mutagens (e.g., chemical mutagens, UV, or ionizing radiation) commonly used. The utility and the improved mutation rate of this system is demonstrated herein by evolving resistance to eight antibiotics in bacterial cells significantly more effectively than when using several other widely used mutagenesis methods. The mutagenesis efficiency conferred by the systems, vectors, and methods provided herein is exemplified herein by an exemplary use of some embodiments of these systems, vectors, and methods to evolve RNA polymerase specificity in bacteriophage under conditions that previously necessitated the use of mutational drift or evolutionary stepping-stones.
• Access to new mutations drives both natural and laboratory evolution. Native biological mutation rates, sometimes referred to herein as “basal mutation rates,” are modest, occurring at frequencies of approximately one mutation per billion replicated DNA bases in most eukaryotes and prokaryotes [1]. Under such native mutation conditions, the time to evolve gene variants that improve organismal fitness is strongly determined by the time required for the mutation to appear [2]. Laboratory evolution methods typically increase basal mutation rates to accelerate the discovery of biomolecules with desired properties on a practical time scale. In addition to mutation rate, the mutational spectrum is also a crucial component of the genetic diversity that fuels evolution. Access to diverse amino acid substitutions during evolution is enhanced by more complete coverage of the 12 possible mutation types [3].
• Several systems to enhance mutational efficiency and broaden mutational spectra have been developed for use in laboratory evolution efforts [4]. In vitro mutagenesis through error-prone PCR, site-saturation mutagenesis, or DNA shuffling has become the standard approach to introduce diversity into genes of interest. Whereas in vitro mutagenesis methods allow for control of mutation rate and mutational spectrum, in vivo mutagenesis methods allow for mutation and selection cycles to be coupled, bypass transformation efficiency bottlenecks that frequently limit the size of gene populations that can be accessed following in vitro mutagenesis, and avoid labor-intensive library creation, cloning, and manipulation steps [5]. The difficulty of tuning mutagenic load and spectrum in live cells, however, has prevented the development of safe, effective in vivo mutagenesis methods that can rival or exceed the efficiency and mutational spectra of state-of-the-art in vitro methods.
• The most commonly used in vivo mutagenesis methods include chemical mutagens, nucleobase analogs, UV light, and hypermutator strains [3]. Chemical mutagens yield narrow mutagenic spectra and are potent human carcinogens. Base analogs offer a safer alternative to chemical mutagenesis, but also exhibit narrow mutational spectra. UV radiation is known to generate a wide mutation spectrum with little sequence preference, but with low potency that is limited by the toxicity of high doses of UV radiation.
• Perhaps the most widespread method for in vivo mutagenesis has been the use of hypermutator strains such as XL-1 Red [6] that have been engineered to have higher mutation rates through the deletion or modification of genes involved in DNA replication and repair. These strains suffer from numerous drawbacks, however, including modest mutational potency (vide infra), moderately biased mutational preference [7], poor transformation efficiency, slow growth rate, and difficulty of modification when additional genetic manipulations are required. In addition, the rate of mutagenesis is not controllable using common hypermutator strains, and mutagenesis generally must be separated from other steps such as selection or screening due to the instability or poor growth of the strain. Loeb and coworkers previously described an elegant system that uses a low-fidelityE. coliDNA Pol I (LF-Pol I) to increase in vivo mutagenesis efficiency with a wide scope of mutational types [8]. Unfortunately, this method is restricted to Pol I temperature-sensitive strains and mutates only vectors carrying ColE1-related origins of replication, with mutation rate being highly dependent on distance from the ColE1 origin [8, 9].
• An in vivo mutagenesis method should offer one or more of the following: (i) a broad mutagenesis spectrum, (ii) a mutation rate that can be very high but is easily modulated by the researcher, and (iii) entirely episomal encoding so that it can be applied to virtually any bacterial strain, including, but not limited to, virtually anyE. colistrain. Some aspects of this disclosure describe the development of general in vivo mutagenesis systems, vectors, and method that meet these criteria.
Mutagenesis Constructs
• Some aspects of this disclosure provide expression constructs encoding gene products that increase the mutation rate in a host cell, e.g., in a bacterial host cell. Expression constructs are sometimes also referred to as vectors. In some embodiments, the expression constructs are plasmids, also referred to herein as mutagenesis plasmids. The use of mutagenesis plasmids in the context of directed evolution has previously been described (e.g., in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference). Some aspects of this disclosure provide improved mutagenesis systems and expression constructs using various combinations of mutagenesis-inducing gene products. The expression constructs provided herein can be used to induce mutagenesis in a target cell, e.g., in a bacterial host cell, at increased rates as compared to conventional mutagenesis agents and methods.
• Some aspects of this disclosure provide expression constructs for modulating the mutation rate of nucleic acids in a cell, e.g., in a bacterial, yeast, or eukaryotic cell. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a gene product that disrupts a proof-reading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway of the bacterial cell, or any combination thereof, wherein the nucleic acid sequence encoding the gene product is under the control of a heterologous promoter.
• Some aspects of this disclosure provide expression constructs encoding a combination of mutagenesis-inducing gene products that allow for robust DNA mutagenesis, e.g., robust chromosomal, episomal, and viral DNA mutagenesis in host cells, e.g., in bacterial host cells. Some exemplary, non-limiting, combinations of mutagenesis-inducing gene products are listed in Table 2 orFIG. 7. The nomenclature of the nucleotides (e.g., genes, transcripts) and proteins referred to herein adheres to the official nomenclature used by the National Center for Biotechnology Information (NCBI). Nucleotide and protein sequences related to the gene symbols listed herein, e.g., in Table 2 orFIG. 7, are known to those of skill in the art. For gene symbols for which publications are indicated (e.g., by superscript numbers or in square brackets), any sequences provided in the publication related to the gene symbol is incorporated herein by reference. In some embodiments, the gene symbols above relate to nucleotide and protein sequences accessible under that gene symbol in any of the National Center for Biotechnology Information (NCBI) databases, for example, in the Nucleotide Reference Sequence (RefSeq) database, release 69 (Jan. 2, 2015), or in the Unigene database available at the time of filing, and any NCBI database entries, e.g., RefSeq database entries for the listed genes in the ResSeq database, release 69, or in the Unigene database at the time of filing, are incorporated herein by reference.
• In some embodiments, a plurality of nucleic acid sequences encoding a gene product that increases the mutation rate in a cell are employed, e.g., combinations of such gene products as shown in Table 2 orFIG. 7. For example, in some embodiments, an a mutagenesis construct provided herein comprises at least two, at least three, at least four, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different nucleic acid sequences encoding a different gene product, e.g., a gene product of Table 2 orFIG. 7. For example, in some embodiments, a mutagenesis expression construct as provided herein may include at least a combination of sequences encoding dnaQ92640, umuD′, umuC, and recA730, and at least one, at least two, at least three, at least four, or at least five additional nucleic acid sequence encoding a different gene product listed in Table 2 orFIG. 7. In some embodiments, a mutagenesis expression construct as provided herein may include at least a combination of sequences encoding dnaQ926, dam, and seqA, and at least one, at least two, at least three, at least four, or at least five additional nucleic acid sequence encoding a different gene product listed in Table 2 orFIG. 7.
• It will be understood that, in some embodiments, the expression constructs provided herein include wild-type sequences of the genes or encoding sequences provided herein, e.g., in Table 2 orFIG. 7, while in other embodiments, one or more of the genes or encoding sequences used may be a functional variant, mutant, fragment, or recombinant form of a wild-type sequence. Such functional variants, mutants, fragments, or recombinant forms include sequences with silent mutations, e.g., codon-optimized sequences, in which nucleotides have been exchanged without altering the sequence of an encoded gene product (e.g., an encoded protein), sequences comprising naturally-occurring polymorphisms, truncations of wild-type sequences that retain the function of the wild-type sequence (e.g., 5′- or 3′ UTR truncations, intron deletions (e.g., cDNAs), and deletions of domains not required for gene product function, and fusions with other sequences, including, e.g., sequences encoding a tag or other gene product. Suitable variants, mutants, fragments, or recombinant forms of wild-type sequences provided herein will be apparent to the skilled artisan based on the instant disclosure.
• In some embodiment, the present disclosure provides expression constructs for modulating the mutagenesis rate in bacterial cells harboring such constructs, also referred to herein as host cells. Such expression constructs can be configured in any suitable manner for introduction into and expression in a bacterial cell, e.g., in the form of a plasmid, cosmid, bacteriophage, or artificial bacterial chromosome. In embodiments, where the expression construct is configured as or comprised in a plasmid, such plasmids are also sometimes referred to herein as “mutagenesis plasmids.” In some embodiments, the bacterial expression constructs provided herein allow for modulation of mutagenesis in bacterial host cells in a strain-independent manner.
• In some embodiments, an expression construct for modulating the mutagenesis rate in a bacterial host cell as provided herein may encode a DNA polymerase lacking a proofreading capability. In some embodiments, the expression construct may encode a gene product involved in the bacterial SOS stress response, for example, a component of a bacterial translesion synthesis polymerase V. In some embodiments, the expression construct may encode a deoxyadenosine methylase. In some embodiments, the expression construct may encode a hemimethylated-GATC binding domain. In some non-limiting embodiments, the expression construct encodes UmuC (a component ofE. colitranslesion synthesis polymerase V), dam (deoxyadenosine methylase), and/or seqA (hemimethylated-GATC binding domain), or any combination thereof.
• In some embodiments, the expression construct for modulating the mutagenesis rate in a bacterial host cell comprises a nucleic acid sequence encoding a gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway of the bacterial cell, or any combination thereof, wherein the nucleic acid sequence encoding the gene product is under the control of a heterologous promoter.
• In some embodiments, the gene product that disrupts a proofreading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product. In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product.
• In some embodiments, the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product. In some embodiments, the recA gene product is a recA730 gene product. In some embodiments, the polB gene product is a polB(D156A) gene product. In some embodiments, the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product. In some embodiments, the mutS gene product is a mutS538, mutS503, or mutSΔN gene product. In some embodiments, the mutL gene product is a mutL705, mutL713, mutL(R261H), or mutL(K307A) gene product. In some embodiments, the mutH gene product is a mutH(E56A), mutH(K79E), or mutH(K116E) gene product. In some embodiments, the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product. In some embodiments, the AID gene product is an AID (7), AID (7.3), AID (7.3.5), AID (7.3.3), AID (7.3.1), or AID (7.3.2) gene product. In some embodiments, the APOBEC gene product is anAPOBEC 1 gene product. In some embodiments, the CDA gene product is a CDA1 gene product. In some embodiments, the MAG gene product is a MAG1 gene product. In some embodiments, the AAG gene product is an AAG(Y127I-H136L) or Δ80-AAG(Y127I-H136L) gene product.
• In some embodiments, the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a umuD′, umuC, or recA730 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a dnaE486, dnaE1026, dnaX36, or dnaX2016 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutS538, mutS503, mutL705, mutL713, mutL(R261H), mutL4K307A), mutH(E56A), mutH(K79E), or mutH(KI 16E) gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR, mutH(E56A), mutL713, mutS503, mutSΔN, dinB, or polB gene product, or any combination thereof.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Dam gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a seqA gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a emrR gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a ugi gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a AID, AID (7), AID (7.3), AID (7.3.5), AID (7.3.3), AID (7.3.1), AID (7.3.2), APOBEC1, or CDA1 gene product, or any combination thereof. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a mutSΔN gene product. In some embodiments, the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, yfjY, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdEF, or nrdAB(P334L)gene product, or any combination thereof.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE74 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE486 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaE1026 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX36 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a dnaX2016 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD12 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD14 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a rpsD16 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a polB(D156A) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a MAG1 gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a AAG(Y127I-H136L) gene product. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a Δ80-AAG(Y127I-H136L) gene product.
• In some embodiments, the expression construct is comprised in a plasmid. In some embodiments, the plasmid comprises a bacterial origin of replication. In some embodiments, the origin of replication is a cloDF13 origin of replication. In some embodiments, the plasmid comprises a nucleic acid sequence encoding a gene product conferring resistance to an antibiotic to a bacterial host cell. In some embodiments, the antibiotic is chloramphenicol, kanamycin, tetracycline, or ampicillin.
• In some embodiments, the expression construct comprises at least one inducible promoter controlling the expression of at least one nucleic acid sequence encoding the gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway, or any combination thereof. In some embodiments, the inducible promoter controls the expression of a nucleic acid encoding a combination of two or more mutagenic gene products, e.g., of any combination listed in Table 2 orFIG. 7. In some embodiments, the expression construct comprises an inducible promoter controlling expression of a nucleic acid sequence encoding a component ofE. colitranslesion synthesis polymerase V, a deoxyadenosine methylase, and/or a hemimethylated-GATC binding domain, or any combination thereof. In some embodiments, the component ofE. colitranslesion synthesis polymerase V is umuC. In some embodiments, the deoxyadenosine methylase is dam. In some embodiments, the hemimethylated-GATC binding domain is seqA. In some embodiments, the nucleic acid sequence controlled by the inducible promoter encodes at least one, at least two, at least three, or at least four additional mutagenic gene products, e.g., as listed in Table 2 orFIG. 7. In some embodiments, the inducible promoter is an arabinose responsive promoter. In some embodiments, the arabinose responsive promoter is a P_BADpromoter. In some embodiments, the expression construct comprises a nucleic acid sequence encoding an arabinose operon regulatory protein. In some embodiments, the arabinose operon regulatory protein is araC. In some embodiments, the nucleic acid sequence encoding the arabinose operon regulatory protein is under the control of a weak promoter. In some embodiments, the weak promoter is a P_Cpromoter.
• In some embodiments, the expression construct comprises at least one codon-optimized nucleic acid sequence encoding a gene product. Methods for codon-optimization and codons preferred by various types of bacterial host cells are well known to those of skill in the art. Some exemplary suitable methods and codons are provided herein, and additional methods and codons will be apparent to the skilled artisan based on the present disclosure. In some embodiments, the codon-optimized nucleic acid sequence comprises at least one codon of a naturally-occurring sequence encoding the gene product that has been replaced with a different codon encoding the same amino acid. In some embodiments, the at least one codon replacing the codon of the naturally occurring nucleic acid sequence corresponds to a tRNA expressed in a bacterial host cell, e.g., in anE. colihost cell. In some embodiments, the at least one codon replacing the codon of the naturally occurring nucleic acid sequence corresponds to a tRNA that is expressed at a higher abundance in the bacterial host cell than the tRNA relating to the naturally occurring tRNA. In some embodiments, the expression construct comprises a nucleic acid sequence encoding a ribosome-binding site, wherein at least one ribosome-binding site encoded by the expression construct is modified as compared to a naturally occurring ribosome binding site. In some embodiments, the modified ribosome binding site exhibits increased ribosome binding as compared to the naturally occurring ribosome binding site. Ribosome-binding sites preferred by ribosomes in various host cells are well known to those of skill in the art.
• In some embodiments, the expression construct comprises a nucleic acid sequence encoding a gene product or a combination of such sequences listed in Table 2 orFIG. 7.
• Some aspects of this disclosure provide plasmids comprising an expression construct as disclosed herein.
• Some aspects of this disclosure provide a cell comprising an expression construct or a plasmid as provided herein. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell further comprises a selection plasmid or an accessory plasmid. In some embodiments, the cell is a host cell for a bacteriophage. In some embodiments, the cell is anE. colicell. In some embodiments, the cell is comprised in a lagoon.
• Some aspects of this disclosure provide vectors and reagents for carrying out continuous evolution processes using the inventive mutagenesis constructs. Such vectors and reagents comprise, for example, selection phage, accessory plasmid, and helper plasmid vectors.
• In some embodiments, a selection phage is provided that comprises a phage genome deficient in at least one gene required for the generation of infectious phage particles and a gene encoding a gene of interest to be evolved.
• For example, in some embodiments, a selection phage as described in PCT Application PCT/US2009/056194, published as WO2010/028347 on Mar. 11, 2010; PCT Application PCT/US2011/066747, published as WO2012/088381 on Jun. 28, 2012; and U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013, the entire contents of each of which are incorporated herein by reference, is provided, that comprises a multiple cloning site for insertion of a nucleic acid sequence encoding a gene to be evolved of interest.
• Such selection phage vectors typically comprise an M13 phage genome deficient in a gene required for the generation of infectious M13 phage particles, for example, a full-length gIII. In some embodiments, the selection phage comprises a phage genome providing all other phage functions required for the phage life cycle except the gene required for generation of infectious phage particles. In some such embodiments, an M13 selection phage is provided that comprises a gI, gII, gIV, gV, gVI, gVII, gVIII, gIX, and a gX gene, but not a full-length gIII. In some embodiments, the selection phage comprises a 3′-fragment of gIII, but no full-length gIII. The 3′-end of gIII comprises a promoter and retaining this promoter activity is beneficial, in some embodiments, for an increased expression of gVI, which is immediately downstream of thegIII 3′-promoter, or a more balanced (wild-type phage-like) ratio of expression levels of the phage genes in the host cell, which, in turn, can lead to more efficient phage production. In some embodiments, the 3′-fragment of gIII gene comprises the 3′-gIII promoter sequence. In some embodiments, the 3′-fragment of gIII comprises the last 180 bp, the last 150 bp, the last 125 bp, the last 100 bp, the last 50 bp, or the last 25 bp of gIII. In some embodiments, the 3′-fragment of gIII comprises the last 180 bp of gIII. In some embodiments, the multiple cloning site for insertion of the gene of interest is located downstream of thegVIII 3′-terminator and upstream of the gIII-3′-promoter.
• Some aspects of this invention provide a vector system for continuous evolution procedures, comprising of a viral vector, for example, a selection phage, comprising a multiple cloning site for insertion of a gene to be evolved, a matching accessory plasmid, and a mutagenesis expression construct as described herein. In some embodiments, a vector system for phage-based continuous directed evolution is provided that comprises (a) a selection phage comprising a multiple cloning site for insertion of a gene of interest to be evolved, wherein the phage genome is deficient in at least one gene required to generate infectious phage; (b) an accessory plasmid comprising the at least one gene required to generate infectious phage particle under the control of a conditional promoter that is activated in response to a desired activity of the gene to be evolved; and (c) a mutagenesis expression construct as provided herein. In some embodiments, the mutagenesis expression construct comprises a nucleic acid sequence encoding a gene or combination of genes as listed in Table 2 orFIG. 7. In some embodiments, the mutagenesis expression construct is a mutagenesis plasmid.
• In some embodiments, the selection phage is an M13 phage as described herein. For example, in some embodiments, the selection phage comprises an M13 genome including all genes required for the generation of phage particles, for example, gI, gII, gIV, gV, gVI, gVII, gVIII, gIX, and gX gene, but not a full-length gIII gene. In some embodiments, the selection phage genome comprises an F1 or an M13 origin of replication. In some embodiments, the selection phage genome comprises a 3′-fragment of gIII gene. In some embodiments, the selection phage comprises a multiple cloning site upstream of thegIII 3′-promoter and downstream of thegVIII 3′-terminator for insertion of a gene of interest.
• The vector system may further comprise a helper phage, wherein the selection phage does not comprise all genes for the generation of infectious phage particles, and wherein the helper phage complements the genome of the selection phage, so that the helper phage genome and the selection phage genome together comprise at least one functional copy of all genes for the generation of phage particles, but are deficient in at least one gene required for the generation of infectious phage particles, which is provided by an accessory plasmid.
Methods
• Some aspects of this disclosure provide methods for modulating the mutation rate in a cell, e.g., in a host cell for bacteriophage. In some embodiments, the method comprises contacting the cell with an expression construct or a plasmid as disclosed herein. In some embodiments, the expression construct comprises an inducible promoter, and the method further comprises culturing the host cell under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, and culturing the host cell under conditions suitable to induce expression from the inducible promoter comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose. In some embodiments, the method results in an at least 10-fold, at least 100-fold, at least 1000-fold, at least 10000-fold, at least 15000-fold, at least 200000-fold, at least 250000-fold, or at least 300000-fold increased mutation rate as compared to the mutation rate in the host cell in the absence of the expression construct.
• Some aspects of this disclosure provide methods for directed evolution using a mutagenesis expression constructs provided herein. In some embodiments, the method comprises (a) contacting a population of host cells comprising a mutagenesis expression construct or plasmid as provided herein with a population of phage vectors comprising a gene to be evolved and deficient in at least one gene for the generation of infectious phage particles, wherein (1) the host cells are amenable to transfer of the vector, (2) the vector allows for expression of the gene to be evolved in the host cell, can be replicated by the host cell, and the replicated vector can transfer into a second host cell; (3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles of (a) in response to the activity of the gene to be evolved, and the level of gene product expression depends on the activity of the gene to be evolved; (b) incubating the population of host cells under conditions allowing for mutation of the gene to be evolved and the transfer of the vector comprising the gene to be evolved from host cell to host cell, wherein host cells are removed from the host cell population, and the population of host cells is replenished with fresh host cells that comprise the expression construct but do not harbor the vector, and (c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene to be evolved.
• In some embodiments, the expression construct comprises an inducible promoter, wherein the incubating of (b) comprises culturing the population of host cells under conditions suitable to induce expression from the inducible promoter. In some embodiments, the inducible promoter is an arabinose-inducible promoter, wherein the incubating of (b) comprises contacting the host cell with an amount of arabinose sufficient to increase expression of the arabinose-inducible promoter by at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least 10000-fold, at least 50000-fold, at least 100000-fold, at least 500000-fold, or at least 1000000-fold as compared to basal expression in the absence of arabinose.
• In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a phage. In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an M13 phage.
• In some embodiments, the at least one gene for the generation of infectious phage particles comprises a sequence encoding a pIII protein. In some embodiments, the at least one gene for the generation of infectious phage particles comprises a full-length gIII gene. In some embodiments, the host cells comprise all phage genes except for the at least one gene for the generation of infectious phage particles in the form of a helper phage. In some embodiments, the phage genes comprised on the helper phage comprise pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, and/or pX.
• In some embodiments, the host cells comprise an accessory plasmid. In some embodiments, the accessory plasmid comprises an expression construct encoding the pIII protein under the control of a promoter that is activated by a gene product encoded by the gene to be evolved. In some embodiments, the host cells comprise the accessory plasmid and together, the helper phage and the accessory plasmid comprise all genes required for the generation of an infectious phage. In some embodiments, the method further comprises a negative selection for undesired activity of the gene to be evolved. In some embodiments, the host cells comprise an expression construct encoding a dominant-negative pIII protein (pIII-neg). In some embodiments, expression of the pIII-neg protein is driven by a promoter the activity of which depends on an undesired function of the gene to be evolved.
• In some embodiments, step (b) comprises incubating the population of host cells for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive life cycles of the viral vector or phage. In some embodiments, the host cells areE. colicells.
• In some embodiments, the host cells are incubated in suspension culture. In some embodiments, the population of host cells is continuously replenished with fresh host cells that do not comprise the vector. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant number of cells in the cell population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector population. In some embodiments, fresh cells are being replenished and cells are being removed from the cell population at a rate resulting in a substantially constant vector, viral, or phage load. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the cells in the cell population. In some embodiments, the rate of fresh cell replenishment and/or the rate of cell removal is adjusted based on quantifying the frequency of host cells harboring the vector and/or of host cells not harboring the vector in the cell population. In some embodiments, the quantifying is by measuring the turbidity of the host cell culture, measuring the host cell density, measuring the wet weight of host cells per culture volume, or by measuring light extinction of the host cell culture.
• In some embodiments, the host cells are exposed to a mutagen. In some embodiments, the mutagen is ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), 0,0-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), or t-butyl hydroperoxide (BHP) (CAS no. 75-91-2).
• In some embodiments, the method comprises a phase of diversifying the population of vector by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the gene to be evolved in the absence of stringent selection for vectors having acquired a gain-of-function mutation in the gene to be evolved. In some embodiments, the method comprises a phase of stringent selection for a mutated replication product of the viral vector encoding an evolved gene.
• One aspect of the PACE directed evolution methods provided herein is the mutation of the initially provided vectors encoding a gene of interest. In some embodiments, the host cells within the flow of cells in which the vector replicates comprise a mutagenesis expression construct as provided herein and are incubated under conditions that increase the natural (or basal) mutation rate. In embodiments, where the mutagenesis expression construct comprises an inducible promoter (e.g., an arabinose-inducible promoter), this may be achieved by contacting the host cells with a compound activating the inducible promoter (e.g., arabinose), in an amount sufficient to increase the mutation rate in the host cells to a desired level, or to increase the activity of the inducible promoter from a basal level to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of its maximum activity, or to fully induce the activity of the inducible promoter.
• In some embodiments, the cells are not exposed to an external mutagen (e.g., a chemical mutagen, UV light, or ionizing radiation) during the PACE processes disclosed herein. In other embodiments, however, the host cells are exposed to an external mutagen in order to further increase the mutation rate in the cells. Typically, the concentration of the mutagen will be chosen to maximize the mutation rate while not being toxic to the host cells during the retention time in the lagoon. Ideally, a mutagen is used at a concentration or level of exposure that induces a desired mutation rate in a given host cell or viral vector population, but is not significantly toxic to the host cells used within the average time frame a host cell is exposed to the mutagen or the time a host cell is present in the host cell flow before being replaced by a fresh host cell. In some embodiments, the mutagen is ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9), or t-butyl hydroperoxide (BHP) (CAS no. 75-91-2). Additional suitable mutagens will be known to those of skill in the art, and the disclosure is not limited in this respect.
• In some embodiments, the mutation rate of the host cells is increased by contacting the cells with a mutagenesis expression construct as provided herein. In some embodiments, the host cells are contacted with a mutagenesis plasmid. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding a mutagenesis-promoting gene product. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding umuC (a component ofE. colitranslesion synthesis polymerase V, e.g., as set forth in GenBank M10107.1), dam (deoxyadenosine methylase, e.g., as set forth in GenBank J01600.1), or seqA (a hemimethylated-GATC binding domain, e.g., as set forth in GenBank U07651.1), or any combination thereof. In some embodiments, the mutagenesis plasmid further comprises a nucleic acid encoding UmuD′, and/or RecA. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding any combination of genes or gene products provided herein, e.g., as provided in Table 2 orFIG. 7.
• In some embodiments, the mutagenesis expression construct comprises an inducible promoter driving expression of at least one mutagenesis-inducing gene or gene product. Suitable inducible promoters are well known to those of skill in the art and include, for example, arabinose-inducible promoters, tetracycline or doxycyclin-inducible promoters, and tamoxifen-inducible promoters. In some embodiments, the host cell population is contacted with an inducer of the inducible promoter in an amount sufficient to effect an increased rate of mutagenesis. For example, in some embodiments, a bacterial host cell population is provided in which the host cells comprise a mutagenesis plasmid in which an expression cassette is controlled by an arabinose-inducible promoter. In some such embodiments, the population of host cells is contacted with the inducer, for example, arabinose, in an amount sufficient to induce a measurably-increased rate of mutation.
• The use of an inducible mutagenesis plasmid allows one to generate a population of fresh, uninfected host cells in the absence of the inducer, thus avoiding an increased rate of mutation in the fresh host cells before they are introduced into the population of cells contacted with the viral vector. Once introduced into this population, however, these cells can then be induced to support an increased rate of mutation, which is particularly useful in some embodiments of continuous evolution. For example, in some embodiments, the host cell comprises a mutagenesis plasmid as described herein, which includes an arabinose-inducible promoter driving expression of umuC, dam, and seqA from a pBAD promoter (see, e.g., Khlebnikov A, Skaug T, Keasling J D.Modulation of gene expression from the arabinose-inducible araBAD promoter. J Ind Microbiol Biotechnol. 2002 Jul.; 29(1):34-7; incorporated herein by reference for disclosure of a pBAD promoter). In some embodiments, the fresh host cells are not exposed to arabinose, which activates expression of the above-identified genes and, thus, increases the rate of mutations in the arabinose-exposed cells, until the host cells reach the lagoon in which the population of selection phage replicates. Accordingly, in some embodiments, the mutation rate in the host cells is normal until they become part of the host cell population in the lagoon, where they are exposed to the inducer (e.g., arabinose) and, thus, to increased mutagenesis. In some embodiments, a method of continuous evolution is provided that includes a phase of diversifying the population of viral vectors by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the viral vector in the absence of stringent selection for the mutated replication product of the viral vector encoding the evolved protein. This is particularly useful in embodiments in which a desired function to be evolved is not merely an increase in an already present function, but the acquisition of a function not present in the gene to be evolved at the outset of the evolution procedure, such as, for example, recognition of a target promoter by a transcription factor showing no binding activity towards the target promoter in the original version of the transcription factor. A step of diversifying the pool of mutated versions of the gene of interest within the population of viral vectors, for example, of phage, allows for an increase in the chance to find a mutation that conveys the desired function, e.g., new substrate specificity in a transcription factor or enzyme.
• In some embodiments, the host cell population is contacted with an agent inducing the expression of an inducible mutagenesis expression construct, such as, for example, arabinose, continuously during a PACE experiment. In other embodiments, the host cell population is contacted with the inducing agent intermittently, creating phases of increased mutagenesis, and accordingly, of increased viral vector diversification. For example, in some embodiments, the host cells are exposed to a concentration of inducing agent sufficient to generate an increased rate of mutagenesis in the gene of interest for about 10%, about 20%, about 30%, about 40%, about 50%, or about 75% of the time. In some embodiments, intermittent exposure to the encoded mutagenesis-increasing gene products can be achieved by using inducible promoters and adding or withdrawing the inducing agent from the host cell culture media during the PACE experiment.
• In some embodiments of the provided methods, (1) the host cells are amenable to transfer of the vector encoding the gene to be evolved; (2) the vector allows for expression of the gene of interest in the host cell, can be replicated by the host cell, and the replicated vector can transfer into a second host cell; and (3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles (a) in response to the activity of the gene of interest, and the level of gene product expression depends on the activity of the gene of interest. The methods of directed evolution provided herein typically comprise (b) incubating the population of host cells under conditions allowing for mutation of the gene of interest, which may include induction of an inducible promoter driving expression of a nucleic acid sequence encoding one or more mutagenic gene products as provided herein, and the transfer of the vector comprising the gene of interest from host cell to host cell. The host cells are removed from the host cell population at a certain rate, e.g., at a rate that results in an average time a host cell remains in the cell population that is shorter than the average time a host cell requires to divide, but long enough for the completion of a life cycle (uptake, replication, and transfer to another host cell) of the vector. The population of host cells is replenished with fresh host cells that do not harbor the vector. In some embodiments, the rate of replenishment with fresh cells substantially matches the rate of removal of cells from the cell population, resulting in a substantially constant cell number or cell density within the cell population. The methods of directed evolution provided herein typically also comprise (c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene of interest.
• In some embodiments, a gene of interest is transferred from host cell to host cell in a manner dependent on the activity of the gene of interest. In some embodiments, the transfer vector is a virus infecting and replicating in the host cells, for example, a bacteriophage or a retroviral vector. In some embodiments, the viral vector is a phage vector infecting bacterial host cells. In some embodiments, the transfer vector is a retroviral vector, for example, a lentiviral vector or a vesicular stomatitis virus vector, infecting human or mouse cells. In some embodiments, the transfer vector is a conjugative plasmid transferred from a donor bacterial cell to a recipient bacterial cell.
• In some embodiments, the nucleic acid vector comprising the gene of interest is a phage, a viral vector, or naked DNA (e.g., a mobilization plasmid). In some embodiments, transfer of the gene of interest from cell to cell is via infection, transfect ion, transduction, conjugation, or uptake of naked DNA, and efficiency of cell-to-cell transfer (e.g., transfer rate) is dependent on an activity of the gene of interest or a mutated version thereof. For example, in some embodiments, the nucleic acid vector is a phage harboring the gene of interest, and the efficiency of phage transfer (via infection) is dependent on the activity of the gene of interest in that a protein for the generation of infectious phage particles (e.g., pIII for M13 phage) is expressed in the host cells only in the presence of a desired activity of the gene of interest.
• Some embodiments provide a continuous evolution system, in which a population of viral vectors, e.g., M13 phage vectors, comprising a gene of interest to be evolved replicates in a flow of host cells that comprise a mutagenesis expression construct provided herein, e.g., a flow through a lagoon, wherein the viral vectors are deficient in a gene encoding a protein that is essential for the generation of infectious viral particles, and wherein that gene is in the host cell under the control of a conditional promoter the activity of which depends on the activity of the gene of interest. Suitable methods, vectors, and reagents for linking the activity of a gene of interest to be evolved to expression of a gene encoding a protein that is essential for the generation of infectious viral particles are described, for example, in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference
• Viral vectors, in which the gene of interest has not acquired a mutation conferring the desired function, will not activate the conditional promoter, or only achieve minimal activation, while any mutation in the gene of interest that confers the desired mutation will result in activation of the conditional promoter. Since the conditional promoter controls an essential protein for the viral life cycle, activation of this promoter directly corresponds to an advantage in viral spread and replication for those vectors that have acquired an advantageous mutation.
• In some embodiments, the viral vector comprising the gene of interest is a phage In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an M13 phage. M13 phages are well known to those in the art and the biology of M13 phages has extensively been studied. A schematic representation of the wild-type M13 genome is provided inFIG. 16. Wild type M13 phage particles comprise a circular, single-stranded genome of approximately 6.4kb. The wild-type genome includes ten genes, gI-gX, which, in turn, encode the ten M13 proteins, pI-pX, respectively, gVIII encodes pVIII, also often referred to as the major structural protein of the phage particles, while gIII encodes pIII, also referred to as the minor coat protein, which is required for infectivity of M13 phage particles.
• The M13 life cycle includes attachment of the phage to the sex pilus of a suitable bacterial host cell via the pIII protein and insertion of the phage genome into the host cell. The circular, single-stranded phage genome is then converted to a circular, double-stranded DNA, also termed the replicative form (RF), from which phage gene transcription is initiated. The wild type M13 genome comprises nine promoters and two transcriptional terminators as well as an origin of replication. This series of promoters provides a gradient of transcription such that the genes nearest the two transcriptional terminators (gVIII and IV) are transcribed at the highest levels. In wild-type M13 phage, transcription of all 10 genes proceeds in same direction. One of the phage-encode proteins, pII, initiates the generation of linear, single-stranded phage genomes in the host cells, which are subsequently circularized, and bound and stabilized by pV. The circularized, single-stranded M13 genomes are then bound by pVIII, while pV is stripped off the genome, which initiates the packaging process. At the end of the packaging process, multiple copies of pIII are attached to wild-type M13 particles, thus generating infectious phage ready to infect another host cell and concluding the life cycle.
• The M13 phage genome can be manipulated, for example, by deleting one or more of the wild type genes, and/or inserting a heterologous nucleic acid construct into the genome. M13 does not have stringent genome size restrictions, and insertions of up to 42 kb have been reported. This allows M13 phage vectors to be used in continuous evolution experiments to evolve genes of interest without imposing a limitation on the length of the gene to be involved.
• The M13 phage has been well characterized and the genomic sequence of M13 has been reported. Representative M13 genomic sequences can be retrieved from public databases, and an exemplary sequence is provided in entry V00604 of the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov):

• Phage M13 genome:
  >gi|56713234|emb|V00604.2| Phage M13 genome
  (SEQ ID NO: 1)
  AACGCTACTACTATTAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATAT
  AGCTAAACAGGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTT
  CGCAGAATTGGGAATCAACTGTTACATGGAATGAAACTTCCAGACACCGTACTTTAGTTGCA
  TATTTAAAACATGTTGAGCTACAGCACCAGATTCAGCAATTAAGCTCTAAGCCATCCGCAAA
  AATGACCTCTTATCAAAAGGAGCAATTAAAGGTACTCTCTAATCCTGACCTGTTGGAGTTTG
  CTTCCGGTCTGGTTCGCTTTGAAGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTT
  CCTCTTAATCTTTTTGATGCAATCCGCTTTGCTTCTGACTATAATAGTGAGGGTAAAGACCT
  GATTTTTGATTTATGGTCATTCTCGTTTTCTGAACTGTTTAAAGCATTTGAGGGGGATTCAA
  TGAATATTTATGACGATTCCGCAGTATTGGACGCTATCCAGTCTAAACATTTTACTATTACC
  CCCTCTGGCAAAACTTCTTTTGCAAAAGCCTCTCGCTATTTTGGTTTTTATCGTCGTCTGGT
  AAACGAGGGTTATGATAGTGTTGCTCTTACTATGCCTCGTAATTCCTTTTGGCGTTATGTAT
  CTGCATTAGTTGAATGTGGTATTCCTAAATCTCAACTGATGAATCTTTCTACCTGTAATAAT
  GTTGTTCCGTTAGTTCGTTTTATTAACGTAGATTTTTCTTCCCAACGTCCTGACTGGTATAA
  TGAGCCAGTTCTTAAAATCGCATAAGGTAATTCACAATGATTAAAGTTGAAATTAAACCATC
  TCAAGCCCAATTTACTACTCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATG
  AGCAGCTTTGTTACGTTGATTTGGGTAATGAATATCCGGTTCTTGTCAAGATTACTCTTGAT
  GAAGGTCAGCCAGCCTATGCGCCTGGTCTGTACACCGTTCATCTGTCCTCTTTCAAAGTTGG
  TCAGTTCGGTTCCCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAG
  GTCGCGGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGC
  GCTTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTCGCCTCTTTCGT
  TTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTTAATGGAAACTTCCTCAT
  GAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTT
  TCGCTGCTGAGGGTGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACC
  GAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAA
  GCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTT
  GGAGCCTTTTTTTTTGGAGATTTTCAACATGAAAAAATTATTATTCGCAATTCCTTTAGTTG
  TTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCCCATACAGAA
  AATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGG
  TTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGTTACGGTA
  CATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGT
  TCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTAT
  TCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACC
  CCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAAT
  AATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCAC
  TGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTT
  ACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCCATTCGTT
  TGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTC
  TGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTG
  GCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAG
  ATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGA
  CGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTG
  GTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCC
  CAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTT
  ACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTAGCGCTGGTAAACCATATG
  AATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATAT
  GTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTA
  ATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGTAAC
  TTTGTTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCTATTT
  CATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGATATT
  AGCGCTCAATTACCCTCTGACTTTGTTCAGGGTGTTCAGTTAATTCTCCCGTCTAATGCGCT
  TCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAA
  AAATCGTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTATTTTGTAACTGGCAAATT
  AGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGGTGCA
  AAATAGCAACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGCTAAA
  ACGCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATATCTGATTTGCTTGCTATTGGGCG
  CGGTAATGATTCCTACGATGAAAATAAAAACGGCTTGCTTGTTCTCGATGAGTGCGGTACTT
  GGTTTAATACCCGTTCTTGGAATGATAAGGAAAGACAGCCGATTATTGATTGGTTTCTACAT
  GCTCGTAAATTAGGATGGGATATTATTTTTCTTGTTCAGGACTTATCTATTGTTGATAAACA
  GGCGCGTTCTGCATTAGCTGAACATGTTGTTTATTGTCGTCGTCTGGACAGAATTACTTTAC
  CTTTTGTCGGTACTTTATATTCTCTTATTACTGGCTCGAAAATGCCTCTGCCTAAATTACAT
  GTTGGCGTTGTTAAATATGGCGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTTATAC
  TGGTAAGAATTTGTATAACGCATATGATACTAAACAGGCTTTTTCTAGTAATTATGATTCCG
  GTGTTTATTCTTATTTAACGCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAATTTA
  GGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGC
  GATTGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTTAAAA
  AGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAAT
  CTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATTTACA
  GAAGCAAGGTTATTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGTAATT
  CAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTTCTTT
  TGCTCAGGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTTGTAACTTGGTATTCAAAGC
  AATCAGGCGAATCCGTTATTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATTCATCT
  GACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTTTTACGTGCTAATAATTTTGA
  TATGGTTGGTTCAATTCCTTCCATAATTCAGAAGTATAATCCAAACAATCAGGATTATATTG
  ATGAATTGCCATCATCTGATAATCAGGAATATGATGATAATTCCGCTCCTTCTGGTGGTTTC
  TTTGTTCCGCAAAATGATAATGTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAAAGGA
  TTTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTAAATCCTCAAATGTATTAT
  CTATTGACGGCTCTAATCTATTAGTTGTTAGTGCACCTAAAGATATTTTAGATAACCTTCCT
  CAATTCCTTTCTACTGTTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATATTTGA
  GGTTCAGCAAGGTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGCGTGGCACTGTTG
  CAGGCGGTGTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGTGGTTCGTTCGGT
  ATTTTTAATGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAAA
  AATATTGTCTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTCTGTTGGCC
  AGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTGCCAATGTAAATAATCCATTT
  CAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGG
  CGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAA
  GTGATGTTATTACTAATCAAAGAAGTATTGCTACAACGGTTAATTTGCGTGATGGACAGACT
  CTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAAGATTCTGGCGTACCGTTCCT
  GTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCCAACGAGGAAAGCA
  CGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGC
  GGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTT
  TCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGG
  GGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTT
  GGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGG
  AGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCG
  GGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCT
  GATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATATTTG
  CTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGAC
  ATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGA
  CCTGATAGCCTTTGTAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTA
  GAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCTTTTGAA
  TCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTA
  TCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTA
  CAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGC
  CTGTATGATTTATTGGATGTT
  GENE II: join (6006..6407,1..831)
  (SEQ ID NO: 3)
  translation = MIDMLVLRLPFIDSLVCSRLSGNDLIAFVDLSKIATLSGMNSLARTVEYHIDGD
  LTVSGLSHPFESLPTHYSGIAFKIYEGSKNFYPCVEIKASPAKVLQGHNVFGTTDLALCSEA
  LLLNFANSLPCLYDLLDVNATTISRIDATFSARAPNENIAKQVIDHLRNVSNGQTKSTRSQN
  WESTVTWNETSRHRTLVAYLKHVELQHQIQQLSSKPSAKMTSYQKEQLKVLSNPDLLEFASG
  LVRFEARIKTRYLKSFGLPLNLFDAIRFASDYNSQGKDLIFDLWSFSFSELFKAFEGDSMNI
  YDDSAVLDAIQSKHFTITPSGKTSFAKASRYFGFYRRLVNEGYDSVALTMPRNSFWRYVSAL
  VECGIPKSQLMNLSTCNNVVPLVRFINVDFSSQRPDWYNEPVLKIA
  GENE X: 496.831
  (SEQ ID NO: 3)
  translation = MNIYDDSAVLDAIQSKHFTITPSGKTSFAKASRYFGFYRRLVNEGYDSVALTMP
  RNSFWRYVSALVECGIPKSQLMNLSTCNNVVPLVRFINVDFSSQRPDWYNEPVLKIA
  GENE V: 843..1106
  (SEQ ID NO: 4)
  translation = MIKVEIKPSQAQFTTRSGVSRQGKPYSLNEQLCYVDLGNEYPVLKITLDEGQP
  AYAPGLYTVHLSSFKVGQFGSLMIDRLRLVPAK
  GENE VII: 1108..1209
  (SEQ ID NO: 5)
  translation = MEQVADFDTIYQAMIQISVVLCFALGIIAGGQR
  GENE IX: 1206..1304
  (SEQ ID NO: 6)
  translation = MSVLVYSFASFVLGWCLRSGITYFTRLMETSS
  GENE VIII: 1301..1522
  (SEQ ID NO: 7)
  translation = MKKSLVLKASVAVATLVPMLSFAAEGDDPAKAAFNSLQASATEYIGYAWAMVVV
  IVGATIGIKLFKKFTSKAS
  GENE III: 1579..2853
  (SEQ ID NO: 8)
  translation = MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGC
  LWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIP
  GYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRPRNRQGALTVYTGTVTQGTD
  PVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSG
  GGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDA
  KGKLDSVATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDNSPLMNNFRQYLP
  SLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES
  GENE VI: 2856..3194
  (SEQ ID NO: 9)
  translation = MPVLLGIPLLLRFLGFLLVTLFGYLLTFLKKGFGKIAIAISLFLALIIGLNSIL
  VGYLSDISAQLPSDFVQGVQLILPSNALPCFYVILSVKAAIFIFDVKQKIVSYLDWDK
  GENE I: 3196..4242
  (SEQ ID NO: 10)
  translation = MAVYFVTGKLGSGKTLVSVGKIQDKIVAGCKIATNLDLRLQNLPQVGRFAKTPR
  VLRIPDKPSISDLLAIGRGNDSYDENKNGLLVLDECGTWFNTRSWNDKERQPIIDWFLHARK
  LGWDIIFLVQDLSIVDKQARSALAEHVVYCRRLDRITLPFVGTLYSLITGSKMPLPKLHVGV
  VKYGDSQLSPTVERWLYTGKNLYNAYDTKQAFSSNYDSGVYSYLTPYLSHGRYFKPLNLGQK
  MKLTKIYLKKFSRVLCLAIGFASAFTYSYITQPKPEVKKVVSQTYDFDKFTIDSSQRLNLSY
  RYVFKDSKGKLINSDDLQKQGYSLTYIDLCTVSIKKGNSNEIVKCN
  GENE IV: 4220..5500
  (SEQ ID NO: 11)
  translation = MKLLNVINFVFLMFVSSSSFAQVIEMNNSPLRDFVTWYSKQSGESVIVSPDVKG
  TVTVYSSDVKPENLRNFFISVLRANNFDMVGSIPSIIQKYNPNNQDYIDELPSSDNQEYDDN
  SAPSGGFFVPQNDNVTQTFKINNVRAKDLIRVVELFVKSNTSKSSNVLSIDGSNLLVVSAPK
  DILDNLPQFLSTVDLPTDQILIEGLIFEVQQGDALDFSFAAGSQRGTVAGGVNTDRLTSVLS
  SAGGSFGIFNGDVLGLSVRALKTNSHSKILSVPRILTLSGQKGSISVGQNVPFITGRVTGES
  ANVNNPFQTIERQNVGISMSVFPVAMAGGNIVLDITSKAOSLSSSTQASDVITNQRSIATTV
  NLRDGQTLLLGGLTDYKNTSQDSGVPFLSKIPLIGLLFSSR3DSNEESTLYVLVKATIVRAL

• Some embodiments of the PACE technology described herein utilize a “selection phage,” a modified phage that comprises a gene of interest to be evolved and lacks a full-length gene encoding a protein required for the generation of infectious phage particles. In some such embodiments, the selection phage serves as the vector that replicates and evolves in the flow of host cells. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a phage gene encoding a protein required for the generation of infectious phage particles, e.g., gI, gII, gIII, gIV, gV, gVI, gVII, gVIII, gIX, or gX, or any combination thereof. For example, some M13 selection phages provided herein comprise a nucleic acid sequence encoding a gene product, e.g., a transcript or a protein, to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a gene encoding a protein required for the generation of infective phage particles, e.g., the gIII gene encoding the pII protein. An exemplary, non-limiting selection plasmid sequence, SP-MCS, comprising a multiple cloning site, into which a nucleic acid sequence encoding a gene product to be evolved can be cloned, is provided below:

• (SEQ ID NO: 30)
  ATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTC
  AGGCAATGACCTGATAGCCTTTGTAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATT
  TATCAGCTAGAACGGTTGAATATCATGTTGATGGTGATTTGACTGTCTCCGGCCTTTCTCAC
  CCTTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAA
  AAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATG
  TTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCT
  TTGCCTTGCCTGTATGATTTATTGGATGTTAACGCTACTACTATTAGTAGAATTGATGCCAC
  CTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGCGAAATG
  TATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATTGGGAATCAACTGTTACATGGAAT
  GAAACTTCCAGACACCGTACTTTAGTTGCATATTTAAAACATGTTGAGCTACAGCACCAGAT
  TCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGAGCAATTAAAGG
  TACTCTCTAATCCTGACCTGTTGGAGTTTGCTTCCGGGCTGGTTCGCTTTGAAGCTCGAATT
  AGAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATGCAATCCGCTTTGC
  TTCTGACTATAATAGTCAGGGTAAAGACCTGATTTTTGATTTATGGTCATTCTCGTTTTCTG
  AACTGTTTAAAGCATTTGAGGGGGATTCAATGAATATTTATGACGATTCCGCAGTATTGGAC
  GCTATCCAGTCTAAACATTTTACTATTACCCCCTCTGGCAAAACTTCTTTTGCAAAAGCCTC
  TCGCTATTTTGGTTTTTATCGTCGTCTGGTAAACGAGGGTTATGATAGTGTTGCTCTTACTA
  TGCCTCGTAATTCCTTTTGGCGTTATGTATCTGCATTAGTTGAATGTGGTATTCCTAAATCT
  CAACTGATGAATCTTTCTACCTGTAATAATGTTGTTCCGTTAGTTCGTTTTATTAACGTAGA
  TTTTTCTTCCCAACGTCCTGACTGGTATAATGAGCCAGTTCTTAAAATCGCATAAGGTAATT
  CACAATGATTAAAGTTGAAATTAAACCATCTCAAGCCCAATTTACTACTCGTTCTGGTGTTT
  CTCGTCAGGGCAAGCCTTATTCACTGAATGAGCAGCTTTGTTACGTTGATTTGGGTAATGAA
  TATCCGGTTCTTGTCAAGATTACTCTTGATGAAGGTCAGCCAGCCTATGCGCCTGGTCTGTA
  CACCGTTCATCTGTCCTCTTTCAAAGTTGGTCAGTTCGGTTCCCTTATGATTGACCGTCTGC
  GCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATCAGGCGAT
  GATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGTATAATCGCTGGGGGTCAAAGATGAG
  TGTTTTAGTGTATTCTTTCGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGT
  ATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAG
  CCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCAAAAGCG
  GCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGT
  TGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCT
  GATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTCGCGCCAGAAGGAGACCA
  AGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCTGGAGA
  TTTTCAACATGCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTAGCGCTGGTAAACCA
  TATGAATTTTCTATTGATTGTGACAAAATGAACTTATTCCGTGGTGTCTTTGCGTTTCTTTT
  ATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGT
  CTTAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGG
  TAACTTTGTTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCT
  ATTTCATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGA
  TATTAGCGCTCAATTACCCTCTGACTTTGTTCAGGGTGTTCAGTTAATTCTCCCGTCTAATG
  CGCTTCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAA
  CAAAAAATCGTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTATTTTGTAACTGGCA
  AATTAGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGG
  TGCAAAATAGCAACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGC
  TAAAACGCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATATCTGATTTGCTTGCTATTG
  GGCGCGGTAATGATTCCTACGATGAAAATAAAAACGGCTTGCTTGTTCTCGATGAGTGCGGT
  ACTTGGTTTAATACCCGTTCTTGGAATGATAAGGAAAGACAGCCGATTATTGATTGGTTTCT
  ACATGCTCGTAAATTAGGATGGGATATTATTTTTCTTGTTCAGGACTTATCTATTGTTGATA
  AACAGGCGCGTTCTGCATTAGCTGAACATGTTGTTTATTGTCGTCGTCTGGACAGAATTACT
  TTACCTTTTGTCGGTACTTTATATTCTCTTATTACTGGCTCGAAAATGCCTCTGCCTAAATT
  ACATGTTGGCGTTGTTAAATATGGCGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTT
  ATACTGGTAAGAATTTGTATAACGCATATGATACTAAACAGGCTTTTTCTAGTAATTATGAT
  TCCGGTGTTTATTCTTATTTAACGCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAA
  TTTAGGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTC
  TTGCGATTGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTT
  AAAAAGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCT
  TAATCTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATT
  TACAGAAGCAAGGTTATTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGT
  AATTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTT
  CTTTTGCTCAGGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTTGTAACTTGGTATTCA
  AAGCAATCAGGCGAATCCGTTATTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATTC
  ATCTGACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTTTTACGTGCAAGTAATT
  TTGATATGGTTGGTTCTAACCCTTCCATTATTCAGAAGTATAATCCAAACAATCAGGATTAT
  ATTGATGAATTGCCATCATCTGATAATCAGGAATATGATGATAATTCCGCTCCTTCTGGTGG
  TTTCTTTGTTCCGCAAAATGATAATGTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAA
  AGGATTTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTAAATCCTCAAATGTA
  TTATCTATTGACGGCTCTAATCTATTAGTTGTTAGTGCACCTAAAGATATTTTAGATAACCT
  TCCTCAATTCCTTTCTACTGTTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATAT
  TTGAGGTTCAGCAAGGTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGCGTGGCACT
  GTTGCAGGCGGTGTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGTGGTTCGTT
  CGGTATTTTTAATGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATT
  CAAAAATATTGTCTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTTTGTT
  GGCCAGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTGCCAATGTAAATAATCC
  ATTTCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGG
  CTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAG
  GCAAGTGATGTTATTACTAATCAAAGAAGTACTGCTACAACGGTTAATTTGCGTGATGGACA
  GACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGT
  TCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAA
  AGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCG
  CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT
  CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA
  TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG
  ATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACG
  TTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTAT
  CTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATG
  AGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATA
  TTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTTATTATCAACCGGGGTACAT

• In some embodiments, the directed evolution methods provided herein comprise an initial or intermittent phase of diversifying the population of vectors by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the gene of interest in the absence of stringent selection or in the absence of any selection for evolved variants of the gene to be evolved that have acquired a desired activity. Such low-stringency selection or no selection periods may be achieved by supporting expression of the gene for the generation of infectious phage particles in the absence of desired activity of the gene to be evolved, for example, by providing an inducible expression construct comprising a gene encoding the respective phage packaging protein under the control of an inducible promoter and incubating under conditions that induce expression of the promoter, e.g., in the presence of the inducing agent. Suitable inducible promoters and inducible expression systems are described herein and in International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; and U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013, the entire contents of each of which are incorporated herein by reference. Additional suitable promoters and inducible gene expression systems will be apparent to those of skill in the art based on the instant disclosure. In some embodiments, the method comprises a phase of stringent selection for a mutated version of the gene to be evolved. If an inducible expression system is used to relieve selective pressure, the stringency of selection can be increased by removing the inducing agent from the population of cells in the lagoon, thus turning expression from the inducible promoter off, so that any expression of the gene required for the generation of infectious phage particles must come from the expression system that depends on the activity of the gene product to be evolved, e.g., the conditional promoter-controlled version of the gene required for the generation of infectious phage particles comprised in an accessory plasmid.
• In some embodiments, the link between desired gene product activity and selective advantage for an encoding phage is provided by an expression system in which at least one gene for the generation of infectious phage particles is expressed in response to the desired activity of the gene to be evolved as described in more detail elsewhere herein. In some embodiments, the at least one gene for the generation of infectious phage particles to another host cell is a gene required for the production of infectious phage particles. In some embodiments, the vector is M13 phage, and the at least one gene for the generation of infectious phage particles comprises a full-length M13 pIII gene. In some embodiments, the host cells comprise an accessory plasmid comprising an expression construct encoding the at least one gene for the generation of infectious phage particles, e.g., the full-length pIII protein, under the control of a conditional promoter that is activated by a desired function of the gene to be evolved.
• In some embodiments, the conditional promoter of the accessory plasmid is a promoter, the transcriptional activity of which can be regulated over a wide range, for example, over 2, 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude by the activating function, for example, the function of a gene of interest. In some embodiments, the conditional promoter has a basal activity that allows for baseline packaging of viral vectors even in the absence of the desired activity of the gene to be evolved or in the presence of only minimal desired activity of the gene to be evolved. This allows for starting a continuous evolution process with a viral vector population comprising versions of the gene of interest that only show minimal activation of the conditional promoter, e.g., of a wild-type version of the gene of interest. In the process of continuous evolution, any mutation in the gene of interest that increases activity of the conditional promoter directly translates into higher expression levels of the gene required for the generation of infectious viral particles in the host cells harboring the vector comprising such a mutation, and, thus, into a competitive advantage over other viral vectors carrying minimally active or loss-of-function versions of the gene of interest.
• One function of the accessory plasmid is to provide a gene for the generation of infectious phage particles under the control of a conditional promoter the activity of which depends on a function of the gene of interest. Accordingly, the accessory plasmid provides a selection mechanism that favors desirable mutations over inconsequential mutations or mutations that are detrimental to the desired function. The stringency of selective pressure imposed by the accessory plasmid in a continuous evolution procedure as provided herein can be modulated. For example, an accessory plasmid may be used at different copy numbers per cell, may comprise a conditional promoter having a base line transcription rate (“leakiness”) that prevents washout of unmutated sequences from the lagoon while still providing a selective advantage to desirable mutations. In some embodiments, an accessory plasmid comprising an expression cassette in which the gene for the generation of infectious phage particles is under the control of an inducible promoter that can be activated by a chemical compound (e.g., a tet-on promoter), allowing for titration of the expression of the gene for the generation of infectious phage particles during a continuous evolution experiment.
• In some embodiments, the use of low copy number accessory plasmids results in an elevated stringency of selection for versions of the gene of interest that activate the conditional promoter on the accessory plasmid, while the use of high copy number accessory plasmids results in a lower stringency of selection. The copy number of an accessory plasmid will depend to a large part on the origin of replication employed. Those of skill in the art will be able to determine suitable origins of replication in order to achieve a desired copy number. The following table lists some non-limiting examples of vectors of different copy numbers and with different origins of replication:

• 	Origin of	Copy
  Plasmids	Replication	number	Class
  pUC vectors	pMB1*	500-700	high copy
  pBluescript ® vectors	ColE1	300-500	high copy
  pGEM ® vectors	pMB1*	300-400	high copy
  pTZ vectors	pMB1*	>1000	high copy
  pBR322 and derivatives	pMB1*	15-20	low copy
  pACYC and derivatives	p15A	10-12	low copy
  pSC101 and derivatives	pSC101	~5	very low copy
  *The pMB1 origin of replication is closely related to that of ColE1 and falls in the same incompatibility group. The high-copy plasmids listed here contain mutated versions of this origin.

• It should be understood that one function of the accessory plasmid, namely to provide a gene for the generation of infectious phage particles under the control of a conditional promoter, the activity of which depends on a function of the gene of interest, can be conferred to a host cell in alternative ways. Such alternatives include, but are not limited to, permanent insertion of a gene construct comprising the conditional promoter and the respective gene into the genome of the host cell, or introducing it into the host cell using a different vector, for example, a phagemid, a cosmid, a phage, a virus, or an artificial chromosome. Additional ways to confer accessory plasmid function to host cells will be evident to those of skill in the art, and the invention is not limited in this respect.
• The sequences of two exemplary, non-limiting accessory plasmids, AP-MCS-A, and AP-MCS-P, respectively, are provided below:

• AP-MCS-A:
  (SEQ ID NO: 31)
  GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAG
  CGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGC
  CGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTA
  GGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA
  TCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAAC
  GTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCA
  AATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTACTCTGCT
  AGCAAGTAAGGCCGACAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCG
  AGCTCGAATTCCCTTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCT
  TTAGTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCCCA
  TACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACT
  ATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGT
  TACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGG
  TGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATA
  CACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAG
  CAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTT
  TCAGAATAATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTC
  AAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTAT
  GACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCC
  ATTCGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCG
  GCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCT
  GAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTA
  TGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTAC
  AGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGT
  TTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTC
  TAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTC
  AATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAA
  CCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCT
  TTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGG
  AGTCTTAATCATGCCAGTTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTT
  TTTTGCCTTGTCGGCCTTACTTGCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA
  ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG
  TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG
  GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT
  CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT
  TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
  CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT
  TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTG
  CGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC
  ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAA
  CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTG
  GCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTT
  GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC
  CGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA
  TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCT
  GAGATAGGTGCCTCACTGATTAAGCATTGGTAAGAACCTCAGATCCTTCCGTGATGGTAACT
  TCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
  AACGTGAGTTTTCGTTCCACTGAGCGTCAGAGAACCTCAGATCCTTCCGTATTTAGCCAGTA
  TGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGC
  TTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAA
  AGCATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTG
  GTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGT
  CTATCTAGTTCAACTTGGAAAATCAACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAA
  TTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCC
  TTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATC
  TCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCT
  CATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTA
  ACTGGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAAC
  TTGGCATAGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCAC
  AGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGA
  TGTTCATCATCTGAGCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGG
  TTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGT
  TAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATACATC
  TCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGATAATTA
  CTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACT
  TGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATA
  TTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCC
  AGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAAC
  GCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAA
  GCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCT
  TTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACT
  ACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGTCACG
  GGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCA
  GCAGTTCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTC
  AGACTGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACT
  AP-MCS-P:
  (SEQ ID NO: 32)
  GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAG
  CGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGC
  CGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTA
  GGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA
  TCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAAC
  GTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCA
  AATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTACTCTGCT
  AGCAAGTAAGGCCGACAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCG
  AGCTCGAATTCCCTTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCT
  TTAGTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAACCCCA
  TACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACT
  ATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGT
  TACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGG
  TGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATA
  CACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAG
  CAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTT
  TCAGAATAATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTC
  AAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTAT
  GACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATCC
  ATTCGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCG
  GCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCT
  GAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTA
  TGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTAC
  AGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGT
  TTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTC
  TAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTC
  AATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAA
  CCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCT
  TTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGG
  AGTCTTAATCATGCCAGTTCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTT
  TTTTGCCTTGTCGGCCTTACTTGCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA
  ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG
  TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTG
  GTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT
  CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT
  TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT
  CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT
  TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTG
  CGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC
  ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAA
  CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTG
  GCGAACTACTTACTCTAGCTTCCCGGCAACAATTGATAGACTGGATGGAGGCGGATAAAGTT
  GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC
  CGGTGAGCGTGGCTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA
  TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCT
  GAGATAGGTGCCTCACTGATTAAGCATTGGTAAGAACCTCAGATCCTTCCGTGATGGTAACT
  TCACTAGTTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT
  AACGTGAGTTTTCGTTCCACTGAGCGTCAGAGAACCTCAGATCCTTCCGTATTTAGCCAGTA
  TGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCATGAGAACGAACCATTGAGATCATGC
  TTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAACTGGTGAGCTGAATTTTTGCAGTTAA
  AGCATCGTGTAGTGTTTTTCTTAGTCCGTTACGTAGGTAGGAATCTGATGTAATGGTTGTTG
  GTATTTTGTCACCATTCATTTTTATCTGGTTGTTCTCAAGTTCGGTTACGAGATCCATTTGT
  CTATCTAGTTCAACTTGGAAAATCAACGTATCAGTCGGGCGGCCTCGCTTATCAACCACCAA
  TTTCATATTGCTGTAAGTGTTTAAATCTTTACTTATTGGTTTCAAAACCCATTGGTTAAGCC
  TTTTAAACTCATGGTAGTTATTTTCAAGCATTAACATGAACTTAAATTCATCAAGGCTAATC
  TCTATATTTGCCTTGTGAGTTTTCTTTTGTGTTAGTTCTTTTAATAACCACTCATAAATCCT
  CATAGAGTATTTGTTTTCAAAAGACTTAACATGTTCCAGATTATATTTTATGAATTTTTTTA
  ACTGGAAAAGATAAGGCAATATCTCTTCACTAAAAACTAATTCTAATTTTTCGCTTGAGAAC
  TTGGCATAGTTTGTCCACTGGAAAATCTCAAAGCCTTTAACCAAAGGATTCCTGATTTCCAC
  AGTTCTCGTCATCAGCTCTCTGGTTGCTTTAGCTAATACACCATAAGCATTTTCCCTACTGA
  TGTTCATCATCTGAGCGTATTGGTTATAAGTGAACGATACCGTCCGTTCTTTCCTTGTAGGG
  TTTTCAATCGTGGGGTTGAGTAGTGCCACACAGCATAAAATTAGCTTGGTTTCATGCTCCGT
  TAAGTCATAGCGACTAATCGCTAGTTCATTTGCTTTGAAAACAACTAATTCAGACATACATC
  TCAATTGGTCTAGGTGATTTTAATCACTATACCAATTGAGATGGGCTAGTCAATGATAATTA
  CTAGTCCTTTTCCTTTGAGTTGTGGGTATCTGTAAATTCTGCTAGACCTTTGCTGGAAAACT
  TGTAAATTCTGCTAGACCCTCTGTAAATTCCGCTAGACCTTTGTGTGTTTTTTTTGTTTATA
  TTCAAGTGGTTATAATTTATAGAATAAAGAAAGAATAAAAAAAGATAAAAAGAATAGATCCC
  AGCCCTGTGTATAACTCACTACTTTAGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAAC
  GCTGTTTGCTCCTCTACAAAACAGACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAA
  GCTCGGGCAAATCGCTGAATATTCCTTTTGTCTCCGACCATCAGGCACCTGAGTCGCTGTCT
  TTTTCGTGACATTCAGTTCGCTGCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACT
  ACAGGCGCCTTTTATGGATTCATGCAAGGAAACTACCCATAATACAAGAAAAGCGCGTCACG
  GGCTTCTCAGGGCGTTTTATGGCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGTTCA
  GCAGTTCCTGCCCTCTGATTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTC
  AGACTGGCTAATGCACCCAGTAAGGCAGCGGTATCATCAACT

• In some embodiments, the gene to be evolved encodes a transcription factor that can directly drive expression from a conditional promoter, resulting in a relatively straightforward linkage of activity of the gene to be evolved to phage packaging efficiency. In embodiments where the gene of interest encodes a gene product that cannot directly drive transcription from a promoter, the linkage of activity of the gene to be evolved to viral particle packaging is provided indirectly, for example, as described in International PCT application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference.
• In some embodiments, the host cells comprise all phage genes except for the at least one gene for the generation of infectious phage particles in the form of a helper phage. In some embodiments, the phage genes on the helper phage include pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, and/or pX. Some exemplary helper phages suitable for use in such embodiments are provided herein, and additional suitable helper phages will be apparent to the skilled artisan based on the instant disclosure. The host cell may also provide phage functions based on expression constructs other than helper phage, for example, expression constructs integrated into the host cell genome or provided on artificial chromosomes or on separate plasmids. One advantage of providing phage functions in the host cell, e.g., by using a helper phage, is that the selection phage encoding the gene of interest can be deficient in genes encoding proteins or other functions provided by the host cell and can, accordingly, carry a longer gene of interest.
• In some embodiments, diversifying the vector population is achieved by providing a flow of host cells that does not select for gain-of-function mutations in the gene of interest for replication, mutagenesis, and propagation of the population of vectors. In some embodiments, the host cells are host cells that express all genes required for the generation of infectious viral particles, for example, bacterial cells that express a complete helper phage, and, thus, do not impose selective pressure on the gene of interest. In other embodiments, the host cells comprise an accessory plasmid comprising a conditional promoter with a baseline activity sufficient to support viral vector propagation even in the absence of significant gain-of-function mutations of the gene of interest. This can be achieved by using a “leaky” conditional promoter, by using a high-copy number accessory plasmid, thus amplifying baseline leakiness, and/or by using a conditional promoter on which the initial version of the gene of interest effects a low level of activity while a desired gain-of-function mutation effects a significantly higher activity.
• Such methods involving host cells of varying selective stringency or varying the selection stringency in other ways as described herein allow for harnessing the power of continuous evolution methods as provided herein for the evolution of functions that are completely absent in the initial version of the gene of interest, for example, for the evolution of enzymes that bind substrates not recognized by the initial enzyme used at the outset of the respective PACE experiments at all.
• In some embodiments, the PACE methods provided herein further comprises a negative selection for undesired activity of the gene to be evolved in addition to the positive selection for a desired activity of the gene to be evolved. Such negative selection methods are useful, for example, in order to maintain enzyme specificity when increasing the efficiency of an enzyme directed towards a specific substrate. This can avoid, for example, the evolution of gene products that show a generally increased activity of the gene to be evolved, including an increased off-target activity, which is often undesired.
• In some embodiments, negative selection is applied during a continuous evolution process as described herein, by penalizing the undesired activities of evolved gene products. This is useful, for example, if the desired evolved gene product is an enzyme with high specificity for a substrate or target site, for example, a transcription factor with altered, but not broadened, specificity. In some embodiments, negative selection of an undesired activity, e.g., off-target activity of the gene to be evolved, is achieved by causing the undesired activity to interfere with pIII production, thus inhibiting the propagation of phage genomes encoding gene products with an undesired activity. In some embodiments, expression of a dominant-negative version of pIII or expression of an antisense RNA complementary to the gIII RBS and/or gIII start codon is linked to the presence of an undesired activity of the gene to be evolved. Suitable negative selection strategies and reagents useful for negative selection, such as dominant-negative versions of M13 pIII, are described herein and in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. application, U.S. Ser. No. 13/922,812, filed Jun. 20, 2013; and U.S. application, U.S. Ser. No. 62/067,194, filed Oct. 22, 2014, the entire contents of each of which are incorporated herein by reference.
• In some embodiments, counter-selection against undesired activity is achieved by linking such undesired activities to the inhibition of phage propagation. In some embodiments, a dual selection strategy is applied during a continuous evolution experiment, in which both positive selection and negative selection constructs are present in the host cells. In some such embodiments, the positive and negative selection constructs are situated on the same plasmid, also referred to as a dual selection accessory plasmid.
• One advantage of using a simultaneous dual selection strategy is that the selection stringency can be fine-tuned based on the activity or expression level of the negative selection construct as compared to the positive selection construct. Another advantage of a dual selection strategy is that the selection is not dependent on the presence or the absence of a desired or an undesired activity, but on the ratio of desired and undesired activities, and, thus, the resulting ratio of pIII and pIII-neg that is incorporated into the respective phage particle.
• For example, in some embodiments, the host cells comprise an expression construct encoding a dominant-negative form of the at least one gene for the generation of infectious phage particles, e.g., a dominant-negative form of the pI protein (pII-neg), under the control of an inducible promoter that is activated by a transcriptional activator other than the transcriptional activator driving the positive selection system. Expression of the dominant-negative form of the gene diminishes or completely negates any selective advantage an evolved phage may exhibit and thus dilutes or eradicates any variants exhibiting undesired activity from the lagoon.
• Some aspects of this invention provide or utilize a dominant negative variant of pIII (pIII-neg). These aspects are based on the recognition that a pIII variant that comprises the two N-terminal domains of pIII and a truncated, termination-incompetent C-terminal domain is not only inactive but is a dominant-negative variant of pill. A pIII variant comprising the two N-terminal domains of pIII and a truncated, termination-incompetent C-terminal domain was described in Bennett, N. J.; Rakonjac, J., Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII.Journal ofMolecular Biology2006, 356 (2), 266-73; the entire contents of which are incorporated herein by reference. The dominant negative property of such pIII variants has been described in more detail in PCT Application PCT/US2011/066747, published as WO2012/088381 on Jun. 28, 2012, the entire contents of which are incorporated herein by reference.
• The pIII-neg variant as provided in some embodiments herein is efficiently incorporated into phage particles, but it does not catalyze the unlocking of the particle for entry during infection, rendering the respective phage noninfectious even if wild type pIII is present in the same phage particle. Accordingly, such pIII-neg variants are useful for devising a negative selection strategy in the context of PACE, for example, by providing an expression construct comprising a nucleic acid sequence encoding a pIII-neg variant under the control of a promoter comprising a recognition motif, the recognition of which is undesired. In other embodiments, pIII-neg is used in a positive selection strategy, for example, by providing an expression construct in which a pII-neg encoding sequence is controlled by a promoter comprising a nuclease target site or a repressor recognition site, the recognition of either one is desired.
• In some embodiments, the vector or phage encoding the gene to be evolved is a filamentous phage, for example, an M13 phage, such as an M13 selection phage as described in more detail elsewhere herein. In some embodiments, the host cells are cells amenable to infection by the filamentous phage, e.g., by M13 phage, such as, for example,E. colicells. In some such embodiments, the gene required for the production of infectious viral particles is the M13 gene III (gIII) encoding the M13 protein III (pIII).
• Typically, the vector/host cell combination is chosen in which the life cycle of the vector is significantly shorter than the average time between cell divisions of the host cell. Average cell division times and vector life cycle times are well known in the art for many cell types and vectors, allowing those of skill in the art to ascertain such host cell/vector combinations. In certain embodiments, host cells are being removed from the population of host cells in which the vector replicates at a rate that results in the average time of a host cell remaining in the host cell population before being removed to be shorter than the average time between cell divisions of the host cells, but to be longer than the average life cycle of the viral vector employed. The result of this is that the host cells, on average, do not have sufficient time to proliferate during their time in the host cell population while the viral vectors do have sufficient time to infect a host cell, replicate in the host cell, and generate new viral particles during the time a host cell remains in the cell population. This assures that the only replicating nucleic acid in the host cell population is the vector encoding the gene to be evolved, and that the host cell genome, the accessory plasmid, or any other nucleic acid constructs cannot acquire mutations allowing for escape from the selective pressure imposed.
• For example, in some embodiments, the average time a host cell remains in the host cell population is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes.
• In some embodiments, the average time a host cell remains in the host cell population depends on how fast the host cells divide and how long infection (or conjugation) requires. In general, the flow rate should be faster than the average time required for cell division, but slow enough to allow viral (or conjugative) propagation. The former will vary, for example, with the media type, and can be delayed by adding cell division inhibitor antibiotics (FtsZ inhibitors inE. coli, etc.). Since the limiting step in continuous evolution is production of the protein required for gene transfer from cell to cell, the flow rate at which the vector washes out will depend on the current activity of the gene(s) of interest. In some embodiments, titrable production of the protein required for the generation of infectious particles, as described herein, can mitigate this problem. In some embodiments, an indicator of phage infection allows computer-controlled optimization of the flow rate for the current activity level in real-time.
• In some embodiments, a PACE experiment according to methods provided herein is run for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles. In certain embodiments, the viral vector is an M13 phage, and the length of a single viral life cycle is about 10-20 minutes.
• In some embodiments, the host cells are contacted with the vector and/or incubated in suspension culture. For example, in some embodiments, bacterial cells are incubated in suspension culture in liquid culture media. Suitable culture media for bacterial suspension culture will be apparent to those of skill in the art, and the invention is not limited in this regard. See, for example,Molecular Cloning: A Laboratory Manual,2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Elizabeth Kutter and Alexander Sulakvelidze:Bacteriophages: Biology and Applications. CRC Press; 1^stedition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski:Bacteriophages: Methods and Protocols, Volume1: Isolation, Characterization, and Interactions(Methods in Molecular Biology) Humana Press; 1^stedition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski:Bacteriophages: Methods and Protocols, Volume2: Molecular and Applied Aspects(Methods in Molecular Biology) Humana Press; 1^stedition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable culture media for bacterial host cell culture).
• Suspension culture typically requires the culture media to be agitated, either continuously or intermittently. This is achieved, in some embodiments, by agitating or stirring the vessel comprising the host cell population. In some embodiments, the outflow of host cells and the inflow of fresh host cells is sufficient to maintain the host cells in suspension. This in particular, if the flow rate of cells into and/or out of the lagoon is high.
• In some embodiments, the flow of cells through the lagoon is regulated to result in an essentially constant number of host cells within the lagoon. In some embodiments, the flow of cells through the lagoon is regulated to result in an essentially constant number of fresh host cells within the lagoon. Typically, the lagoon will hold host cells in liquid media, for example, cells in suspension in a culture media. However, lagoons in which adherent host cells are cultured on a solid support, such as on beads, membranes, or appropriate cell culture surfaces are also envisioned. The lagoon may comprise additional features, such as a stirrer or agitator for stirring or agitating the culture media, a cell densitometer for measuring cell density in the lagoon, one or more pumps for pumping fresh host cells into the culture vessel and/or for removing host cells from the culture vessel, a thermometer and/or thermocontroller for adjusting the culture temperature, as well as sensors for measuring pH, osmolarity, oxygenation, and other parameters of the culture media. The lagoon may also comprise an inflow connected to a holding vessel comprising a mutagen or a transcriptional inducer of a conditional gene expression system, such as the arabinose-inducible expression system of the mutagenesis plasmid described in more detail elsewhere herein.
• In some embodiments, the host cell population is continuously replenished with fresh, uninfected host cells. In some embodiments, this is accomplished by a steady stream of fresh host cells into the population of host cells. In other embodiments, however, the inflow of fresh host cells into the lagoon is semi-continuous or intermittent (e.g., batch-fed). In some embodiments, the rate of fresh host cell inflow into the cell population is such that the rate of removal of cells from the host cell population is compensated. In some embodiments, the result of this cell flow compensation is that the number of cells in the cell population is substantially constant over the time of the continuous evolution procedure. In some embodiments, the portion of fresh, uninfected cells in the cell population is substantially constant over the time of the continuous evolution procedure. For example, in some embodiments, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, or about 90% of the cells in the host cell population are not infected by virus. In general, the faster the flow rate of host cells is, the smaller the portion of cells in the host cell population that are infected will be. However, faster flow rates allow for more transfer cycles, e.g., viral life cycles, and, thus, for more generations of evolved vectors in a given period of time, while slower flow rates result in a larger portion of infected host cells in the host cell population and therefore a larger library size at the cost of slower evolution. In some embodiments, the range of effective flow rates is invariably bounded by the cell division time on the slow end and vector washout on the high end In some embodiments, the viral load, for example, as measured in infectious viral particles per volume of cell culture media is substantially constant over the time of the continuous evolution procedure.
• Typically, the fresh host cells introduced into the lagoon comprise a mutagenesis expression construct as provided herein, a selection system, e.g., an accessory plasmid encoding the at least one gene for the generation of infectious phage particles, and an expression construct providing other phage functions, such as, for example, a helper phage. In some embodiments, however, the host cells may not comprise an expression construct providing other phage functions, such as, for example, a helper phage, and those functions are provided to the host cells in some other way, e.g., as part of the selection phage. In some embodiments, the host cells are generated by contacting an uninfected host cell with the relevant vectors, for example, a vector comprising a mutagenesis expression construct as provided herein, an accessory plasmid, and, if desired, a helper phage, and growing an amount of host cells sufficient for the replenishment of the host cell population in a continuous evolution experiment. Methods for the introduction of plasmids and other gene constructs into host cells are well known to those of skill in the art and the invention is not limited in this respect. For bacterial host cells, such methods include, but are not limited to electroporation and heat-shock of competent cells. In some embodiments, the accessory plasmid comprises a selection marker, for example, an antibiotic resistance marker, and the fresh host cells are grown in the presence of the respective antibiotic to ensure the presence of the plasmid in the host cells. Where multiple plasmids are present, different markers are typically used. Such selection markers and their use in cell culture are known to those of skill in the art, and the invention is not limited in this respect.
• In some embodiments, the host cell population in a continuous evolution experiment is replenished with fresh host cells growing in a parallel, continuous culture. In some embodiments, the cell density of the host cells in the host cell population contacted with the viral vector and the density of the fresh host cell population is substantially the same.
• Typically, the cells being removed from the cell population contacted with the vector comprise cells that are infected with the vector and uninfected cells. In some embodiments, cells are being removed from the cell populations continuously, for example, by effecting a continuous outflow of the cells from the population. In other embodiments, cells are removed semi-continuously or intermittently from the population. In some embodiments, the replenishment of fresh cells will match the mode of removal of cells from the cell population, for example, if cells are continuously removed, fresh cells will be continuously introduced. However, in some embodiments, the modes of replenishment and removal may be mismatched, for example, a cell population may be continuously replenished with fresh cells, and cells may be removed semi-continuously or in batches.
• In some embodiments, the rate of fresh host cell replenishment and/or the rate of host cell removal is adjusted based on quantifying the host cells in the cell population. For example, in some embodiments, the turbidity of culture media comprising the host cell population is monitored and, if the turbidity falls below a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect an increase in the number of host cells in the population, as manifested by increased cell culture turbidity. In other embodiments, if the turbidity rises above a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect a decrease in the number of host cells in the population, as manifested by decreased cell culture turbidity. Maintaining the density of host cells in the host cell population within a specific density range ensures that enough host cells are available as hosts for the evolving viral vector population, and avoids the depletion of nutrients at the cost of viral packaging and the accumulation of cell-originated toxins from overcrowding the culture.
• In some embodiments, the cell density in the host cell population and/or the fresh host cell density in the inflow is about 10²cells/ml to about 10¹²cells/ml. In some embodiments, the host cell density is about 10²cells/ml, about 10³cells/ml, about 10⁴cells/ml, about 10⁵cells/ml, about 5·10 cells/ml, about 10⁶cells/ml, about 5·10⁶cells/ml, about 10⁷cells/ml, about 5·10⁷cells/ml, about 10⁸cells/ml, about 5·10⁸cells/ml, about 10⁹cells/ml, about 5·10⁹cells/ml, about 10¹⁰cells/ml, or about 5·10¹⁰cells/ml. In some embodiments, the host cell density is more than about 10¹⁰cells/ml.
• The PACE methods provided herein are typically carried out in a lagoon. Suitable lagoons and other laboratory equipment for carrying out PACE methods as provided herein have been described in detail elsewhere. See, for example, International PCT Application, PCT/US2011/066747, published as WO2012/088381 on Jun. 28, 2012, the entire contents of which are incorporated herein by reference. In some embodiments, the lagoon comprises a cell culture vessel comprising an actively replicating population of vectors, for example, phage vectors comprising a gene of interest, and a population of host cells, for example, bacterial host cells. In some embodiments, the lagoon comprises an inflow for the introduction of fresh host cells into the lagoon and an outflow for the removal of host cells from the lagoon. In some embodiments, the inflow is connected to a turbidostat comprising a culture of fresh host cells. In some embodiments, the outflow is connected to a waste vessel, or a sink. In some embodiments, the lagoon further comprises an inflow for the introduction of a mutagen into the lagoon. In some embodiments that inflow is connected to a vessel holding a solution of the mutagen. In some embodiments, the lagoon comprises an inflow for the introduction of an inducer of gene expression into the lagoon, for example, of an inducer activating an inducible promoter within the host cells that drives expression of a gene promoting mutagenesis (e.g., as part of a mutagenesis plasmid), as described in more detail elsewhere herein. In some embodiments, that inflow is connected to a vessel comprising a solution of the inducer, for example, a solution of arabinose.
• In some embodiments, the lagoon comprises a controller for regulation of the inflow and outflow rates of the host cells, the inflow of the mutagen, and/or the inflow of the inducer. In some embodiments, a visual indicator of phage presence, for example, a fluorescent marker, is tracked and used to govern the flow rate, keeping the total infected population constant. In some embodiments, the visual marker is a fluorescent protein encoded by the phage genome, or an enzyme encoded by the phage genome that, once expressed in the host cells, results in a visually detectable change in the host cells. In some embodiments, the visual tracking of infected cells is used to adjust a flow rate to keep the system flowing as fast as possible without risk of vector washout.
• In some embodiments, the controller regulates the rate of inflow of fresh host cells into the lagoon to be substantially the same (volume/volume) as the rate of outflow from the lagoon. In some embodiments, the rate of inflow of fresh host cells into and/or the rate of outflow of host cells from the lagoon is regulated to be substantially constant over the time of a continuous evolution experiment. In some embodiments, the rate of inflow and/or the rate of outflow is from about 0.1 lagoon volumes per hour to about 25 lagoon volumes per hour. In some embodiments, the rate of inflow and/or the rate of outflow is approximately 0.1 lagoon volumes per hour (lv/h), approximately 0.2 lv/h, approximately 0.25 lv/h, approximately 0.3 lv/h, approximately 0.4 lv/h, approximately 0.5 lv/h, approximately 0.6 lv/h, approximately 0.7 lv/h, approximately 0.75 lv/h, approximately 0.8 lv/h, approximately 0.9 lv/h, approximately 1 lv/h, approximately 2 lv/h, approximately 2.5 lv/h, approximately 3 lv/h, approximately 4 lv/h, approximately 5 lv/h, approximately 7.5 lv/h, approximately 10 lv/h, or more than 10 lv/h.
• In some embodiments, the inflow and outflow rates are controlled based on a quantitative assessment of the population of host cells in the lagoon, for example, by measuring the cell number, cell density, wet biomass weight per volume, turbidity, or cell growth rate. In some embodiments, the lagoon inflow and/or outflow rate is controlled to maintain a host cell density of from about 10²cells/ml to about 10¹²cells/ml in the lagoon. In some embodiments, the inflow and/or outflow rate is controlled to maintain a host cell density of about 10²cells/ml, about 10³cells/ml, about 10⁴cells/ml, about 10⁵cells/ml, about 5×10⁵cells/ml, about 10⁶cells/ml, about 5×10⁶cells/ml, about 10⁷cells/ml, about 5×10⁷cells/ml, about 10⁸cells/ml, about 5×10⁸cells/ml, about 10⁹cells/ml, about 5×10⁹cells/ml, about 10¹⁰cells/ml, about 5×10¹⁰cells/ml, or more than 5×10¹⁰cells/ml, in the lagoon. In some embodiments, the density of fresh host cells in the turbidostat and the density of host cells in the lagoon are substantially identical.
• In some embodiments, the lagoon inflow and outflow rates are controlled to maintain a substantially constant number of host cells in the lagoon. In some embodiments, the inflow and outflow rates are controlled to maintain a substantially constant frequency of fresh host cells in the lagoon. In some embodiments, the population of host cells is continuously replenished with fresh host cells that are not infected by the phage. In some embodiments, the replenishment is semi-continuous or by batch-feeding fresh cells into the cell population.
• In some embodiments, the lagoon volume is from approximately 1 ml to approximately 100 l, for example, the lagoon volume is approximately 1 ml, approximately 10 ml, approximately 50 ml, approximately 100 ml, approximately 200 ml, approximately 250 ml, approximately 500 ml, approximately 750 ml, approximately 11, approximately 2 l, approximately 2.5 l, approximately 3 l, approximately 4 l, approximately 5 l, approximately 10 l, approximately 20 l, approximately 50 l, approximately 75 l, approximately 100 l, approximately 1 ml-10 ml, approximately 10 ml-50 ml, approximately 50 ml-100 ml, approximately 100 ml-250 ml, approximately 250 ml-500 ml, approximately 500 ml-1 l, approximately 1 l-2 l, approximately 2 l-5 l, approximately 5 l-10 l, approximately 10 l-50 l, approximately 50 l-100 l, or more than 100 l.
• In some embodiments, the lagoon and/or the turbidostat further comprises a heater and a thermostat controlling the temperature. In some embodiments, the temperature in the lagoon and/or the turbidostat is controlled to be from about 4° C. to about 55° C., preferably from about 25° C. to about 39° C., for example, about 37° C.
• In some embodiments, the inflow rate and/or the outflow rate is controlled to allow for the incubation and replenishment of the population of host cells for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive vector or phage life cycles. In some embodiments, the time sufficient for one phage life cycle is about 10, 15, 20, 25, or 30 minutes.
• Therefore, in some embodiments, the time of the entire evolution procedure is about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 50 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about two weeks, about 3 weeks, about 4 weeks, or about 5 weeks.
• In some embodiments, a PACE method as provided herein is performed in a suitable apparatus as described herein. For example, in some embodiments, the apparatus comprises a lagoon that is connected to a turbidostat comprising a host cell as described herein. In some embodiments, the host cell is anE. colihost cell. In some embodiments, the host cell comprises a mutagenesis expression construct as provided herein, an accessory plasmid as described herein, and, optionally, a helper plasmid as described herein, or any combination thereof. In some embodiments, the lagoon further comprises a selection phage as described herein, for example, a selection phage encoding a gene of interest. In some embodiments, the lagoon is connected to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose. In some embodiments, the host cells areE. colicells comprising the F′ plasmid, for example, cells of the genotype F′proA⁺B⁺Δ(lacIZY) zzf::Tn10(TetR)/endA 1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻.
• For example, in some embodiments, a PACE method as provided herein is carried out in an apparatus comprising a lagoon of about 100 ml, or about 1 l volume, wherein the lagoon is connected to a turbidostat of about 0.5 l, 1 l, or 3 l volume, and to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose, wherein the lagoon and the turbidostat comprise a suspension culture ofE. colicells at a concentration of about 5×10⁸cells/ml. In some embodiments, the flow of cells through the lagoon is regulated to about 3 lagoon volumes per hour. In some embodiments, cells are removed from the lagoon by continuous pumping, for example, by using a waste needle set at a height of the lagoon vessel that corresponds to a desired volume of fluid (e.g., about 100 ml, in the lagoon. In some embodiments, the host cells areE. colicells comprising the F′ plasmid, for example, cells of the genotype F′proA⁺B⁺Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻.
• In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, umuD′, umuC, and recA730, and is referred to as “MP1”. In some embodiments, a mutagenesis plasmid (MP) comprises araC and dnaQ926, and is referred to as “MP2”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, and dam, and is referred to as “MP3”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, dam, and seqA, and is referred to as “MP4”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1, and is referred to as “MP5”. In some embodiments, a mutagenesis plasmid (MP) comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1, and is referred to as “MP6”. In some embodiments, a mutagenesis plasmid comprises a sequence represented by any one of SEQ ID NO: 43-123. In some embodiments, a mutagenesis plasmid consists of a sequence represented by any one of SEQ ID NO: 43-123.
Modulation of Selection Stringency
• Provided herein is a method for modulating the selection stringency in viral-assisted continuous evolution experiments. In some embodiments, the selection stringency is modulated by regulating the expression of a gene required for the generation of infectious viral particles (e.g., infectious phages). Generally, the gene required for the generation of infectious viral particles is on an accessory plasmid (AP) or on a drift plasmid (DP).
• A drift plasmid allows evolutionary drift to take place to evolve weakly active or inactive gene variants. The expression of a gene required for the generation of infectious viral particles produces a protein required for the generation of infectious viral particles. In some embodiments, the gene required for the generation of infectious viral particles is gene III, which expresses the protein pIII needed to generate infections phage. In some embodiments, the modulation of the selection stringency is independent of the desired activity to be evolved. In some embodiments, regulation of the expression of the gene required for the generation of infectious viral particles is under the control of a small molecule inducible promoter (i.e., chemically-regulated promoters) and therefore, is dependent on the concentration of a small molecule. Examples of small molecule inducible promoters are known in the scientific literature (see, e.g., Yamamoto et. al., 2001, Neurobiology of Disease, 8: 923-932). Non-limiting examples of small molecule inducible promoters include lux promoters (e.g. P_luxfrom vibrio fishceri induced by N-(3-oxohexanoyl)-L-homoserine lactone (OHHL)); alcohol-regulated promoters (e.g., alcohol dehydrogenase I promoter (alcA), lac promoter (e.g., P_lac), arabinose-inducible promoters (e.g., P_ara), tetracycline-inducible promoters (e.g., P_tet), steroid-inducible promoters, and tamoxifen-inducible promoters. In some embodiments, the small molecule inducible promoter is a TetA promoter (P_tet). In some embodiments, the small molecule is tetracycline or tetracycline analogs. In some embodiments, the small molecule is anhydrotetracycline (ATc). In some embodiments, the small molecule is doxycycline. In some embodiments, the host cell drift promoter is partly a tetracycline-inducible promoter (P_tet), which drives expression of the TetR repressor and TetA, the protein that pumps tetracycline out of the cell. In the absence of tetracycline or its analogs, TetR binds to the TetR operator sites and prevents transcription. In the presence of tetracycline or its analogs. TetR binds to tetracycline or a tetracycline analog, which induces a conformational change, making it unable to interact with the operator, so that target gene expression can occur.
• In some embodiments, the host cell becomes viral infection-resistant prior to encountering the viral particle, thereby preventing viral propagation. For example, low levels of pill, such as the levels expressed at the beginning of a PACE experiment, have been shown to render cells resistant to filamentous phage infection.¹⁰Accordingly, for situations where low levels of the protein (e.g., pIII) required for the generation of infectious viral particles renders host cells resistant to viral infection, it may be desirable to make the expression of the protein (e.g., pIII) required for the generation of infectious viral particles to be dependent on the condition that there be a prior viral infection of the host cells. In some embodiments, anE. coliphage shock promoter (P_psp) is used to require prior viral infection. Transcription from P_pspis induced by infection with filamentous phage via a pIV-dependent signaling cascade¹¹or by overexpression of a plasmid-encoded phage pIV gene.
• To produce a system in which protein expression requires both the presence of the small molecule and prior viral infection, provided herein is a drift promoter. In some embodiments, the drift promoter is located on a drift plasmid in the host cell. In some embodiments, the drift promoter is produced from a P_pspvariant with a TetR operator installed at a position to disrupt either PspF orE. coliRNA polymerase binding. In some embodiments, the TetR operator is placed adjacent to the +1 transcription initiation site to produce a host cell drift promoter called P_psp-tetwhich is induced only with the combination of phage infection and ATc. In some embodiments, the P_psp-tetis placed upstream of the gene encoding the pIII protein. In some embodiments, propagation of the viral vector (e.g., SP) proceeds without activity-dependent gene III expression. In some embodiments, SPs can propagate in a small-molecule-dependent, activity-independent manner using the host cell drft promoter. In some embodiments, the drift promoter is produced from one that is activated upon pspF release after phage infection. In some embodiments, the host cell drift promoter is produced from another promoter such as ones upstream of pal or hyfR.
• In some embodiments, a drift plasmid (DP) comprises an expression construct in which a drift promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII), and an expression construct comprising a sequence encoding one or more gene product(s) that increase(s) the mutation rate in a host cell, e.g., in a bacterial host cell. The one or more gene product(s) that increase(s) the mutation rate in a host cell is, in some embodiments, araC, dnaQ926, umuD′, umuC, recA730, dam, seqA, emrR, PBS2, UGI, or pmCDA1, or any combination thereof. In some embodiments, a drift plasmid (DP) comprises an expression construct in which a drift promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII), wherein the expression construct is on the same plasmid as a mutagenesis expression construct provided herein, e.g., a mutagenesis expression construct provided in the context of the mutagenesis plasmids provided herein.
• In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, umuD′, umuC, recA730, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP1”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP1 plasmid is shown inFIG. 20, and is represented by SEQ ID NO: 27.
• In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP2”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP2 plasmid is shown inFIG. 21, and is represented by SEQ ID NO: 28.
• In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP3”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP3 plasmid is shown inFIG. 22, and is represented by SEQ ID NO: 29.
• In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, seqA, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP4”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP4 plasmid is shown inFIG. 23, and is represented by SEQ ID NO: 33.
• In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, seqA, ugi and pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP5”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP5 plasmid is shown inFIG. 24, and is represented by SEQ ID NO: 34.
• In some embodiments, a drift plasmid (DP) comprises araC, dnaQ926, dam, seqA, emrR, ugi and pmCDA1, and an anhydrotetracycline (ATc)-dependent drift promoter, and is referred to as “DP6”. In some embodiments, the ATc-dependent promoter drives expression of a gene required for the generation of infectious viral particles (e.g., gIII). One embodiment of a DP6 plasmid is shown inFIG. 25, and is represented by SEQ ID NO: 35.
• In some embodiments, provided is a method of tuning the selection stringency in continuous directed evolution methods. For example, to tune the selection stringency, a host cell can use the following plasmids: an activity-dependent AP, such as a P_T7-gene III AP, in which gene II is controlled by an activity-dependent promoter, and a drift plasmid (DP) with a host cell drift promoter-gene III cassette, such as a P_psp-tetgene III. In some embodiments, the AP additionally contains a reporter gene such as a luciferase gene. In some embodiments, the selection stringency is inversely proportional to the concentration of the small molecule used. In some embodiments, low selection stringency conditions are used. For example, saturating amounts of a small molecule inducer (e.g., ATc) allows the P_psp-tet-gene III cassette in the DP to provide sufficient pII to maximize phage propagation, regardless of the SP-encoded property (such as activity), thus enabling genetic drift of the SP (low stringency). In some embodiments, an intermediate selection stringency is used. For example, at intermediate concentrations of a small molecule inducer (e.g., ATc), SPs encoding active library members have a replicative advantage over an SP encoding a weakly active/inactive variant by inducing additional pIII expression from an activity-dependent manner. An intermediate concentration is determined by sampling a number of concentrations of the small molecule by using a plasmid the carries the native P_tetpromoter driving bacterial luciferase. Intermediate concentrations are typically considered those around the inflection point of a sigmoidal graph. In some embodiments, high selection stringency conditions are used. For example, a zero or low amount of a small molecule inducer (e.g., ATc) allows the selection stringency to be determined by the activity-dependent AP with no assistance from the P_psp-tet-gene III cassette (high stringency).
• In some embodiments, the evolution experiments uses a ratio of SPs with active starting genetic libraries to SPs with weakly active/inactive starting genetic libraries of about 1:1, 1:5, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, 1:120, 1:60, or 1:200. In some embodiments, the ratio of SPs with active to weakly active/inactive starting libraries is 1:100. In some embodiments, phage population is generally followed over time using a detectable label or directly via standard techniques. For example, the phage population can be followed using a combination of restriction endonuclease digests and/or real-time measurements of luminescence monitoring of promoter transcriptional activity (e.g., P_T7transcriptional activity), as further described herein. Additional methods are PCR, plaque assays, analysis by gel electrophoresis, or analytical digestion. In some embodiments, an accessory plasmid carrying the gene III and a gene encoding a co-expressed reporter fluorescent protein (such as the luciferase gene, GFP, or other fluorescent protein described herein) under the control of a conditional promoter (such as a P_T7or P_T3) would produce luminescence from the translated luciferase when there is promoter transcriptional activity.
• Selection stringency modulation can be used at any point in the continuous evolution process. In some embodiments, selection stringency modulation is used towards the end of the continuous evolution process. In some embodiments, selection stringency modulation is used towards the beginning of the continuous evolution process. In some embodiments, the selection stringency modulation is combined with negative selection.
• In an embodiment, provided is a method for modulating the selection stringency during viral-assisted evolution of a gene product, the method comprising: (a) introducing host cells into a lagoon, wherein the host cell comprises a low selection stringency plasmid and a high selection stringency plasmid, wherein the low selection stringency plasmid comprises a viral gene required to package the selection viral vector into an infectious viral particles, wherein at least one gene required to package the selection viral vector into an infectious viral particles is expressed in response to the a concentration of a small molecule, and wherein the high selection stringency plasmid comprises a second copy of the viral gene required to package the selection viral vector into the infectious viral particles, wherein at least one viral gene required to package the selection viral vector into an infectious viral particles is expressed in response to a desired activity property of a gene product encoded by the gene to be evolved or an evolution product thereof; (b) introducing a selection viral vector comprising a gene to be evolved into a flow of host cells through a lagoon, wherein the gene to be evolved produces an active gene product or a weakly active or inactive gene product, wherein the active gene product has an activity that drives the expression of the viral gene required to package the selection viral vector into infectious viral particles in the high selection stringency plasmid and wherein the weakly active or inactive gene product has a relatively lower activity than the activity of the active gene product; and (c) mutating the gene to be evolved within the flow of host cells, wherein the host cells are introduced through the lagoon at a flow rate that is faster than the replication rate of the host cells and slower than the replication rate of the virus thereby permitting replication of the selection viral vector in the lagoon. In an embodiment, the host cells are fed from a chemostat into the lagoon.
• In some embodiments, the method further comprising isolating the selection viral vector comprising an evolved product from the flow of cells and determining one or more properties of the evolved product. In some embodiments, the low selection stringency plasmid contains a drift promoter that is activated by a concentration of a small molecule inducer and/or prior viral infection. In one embodiment, the high selection stringency plasmid contains a promoter that is activated by a desired property of a gene product encoded by the gene to be evolved or an evolution product thereof. In yet another embodiment, the low selection stringency plasmid comprises a mutagenesis cassette under the control of a small-molecule inducible promoter. In another embodiment, the low selection stringency plasmid allows a high level of evolutionary drift to occur when the drift promoter is activated in response to a concentration of a small molecule inducer and/or prior viral infection. In some embodiments, the high selection stringency plasmid allows a low level of evolutionary drift to occur when the promoter is activated in response to a desired activity property of a gene product encoded by the gene to be evolved or an evolution product thereof.
• In some embodiments, the property of the gene to be evolved originated from a weakly active or inactive starting gene. In some embodiments, the property of the gene to be evolved originated from an active starting gene. In an embodiment, the high selection stringency comprises a T7 promoter. In another embodiment, the low selection stringency comprises a drift promoter that is activated by a small-molecule inducer and/or prior viral infection. In some embodiments, the drift promoter is a Ppsp-tet promoter.
• In some embodiments, the method of modulating the selection stringency further comprises the use of negative selection and/or positive selection.
Host Cells
• Some aspects of this invention relate to host cells for continuous evolution processes as described herein. In some embodiments, a host cell is provided that comprises a mutagenesis expression construct as provided herein. In some embodiments, the host cell further comprises additional plasmids or constructs for carrying out a PACE process, e.g., a selection system comprising at least one viral gene encoding a protein required for the generation of infectious viral particles under the control of a conditional promoter the activity of which depends on a desired function of a gene to be evolved. For example, some embodiments provide host cells for phage-assisted continuous evolution processes, wherein the host cell comprises an accessory plasmid comprising a gene required for the generation of infectious phage particles, for example, M13 gIII, under the control of a conditional promoter, as described herein. In some embodiments, the host cell further provides any phage functions that are not contained in the selection phage, e.g., in the form of a helper phage. In some embodiments, the host cell provided further comprises an expression construct comprising a gene encoding a mutagenesis-inducing protein, for example, a mutagenesis plasmid as provided herein.
• In some embodiments, modified viral vectors are used in continuous evolution processes as provided herein. In some embodiments, such modified viral vectors lack a gene required for the generation of infectious viral particles. In some such embodiments, a suitable host cell is a cell comprising the gene required for the generation of infectious viral particles, for example, under the control of a constitutive or a conditional promoter (e.g., in the form of an accessory plasmid, as described herein). In some embodiments, the viral vector used lacks a plurality of viral genes. In some such embodiments, a suitable host cell is a cell that comprises a helper construct providing the viral genes required for the generation of infectious viral particles. A cell is not required to actually support the life cycle of a viral vector used in the methods provided herein. For example, a cell comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter may not support the life cycle of a viral vector that does not comprise a gene of interest able to activate the promoter, but it is still a suitable host cell for such a viral vector.
• In some embodiments, the host cell is a prokaryotic cell, for example, a bacterial cell. In some embodiments, the host cell is anE. colicell. In some embodiments, the host cell is a eukaryotic cell, for example, a yeast cell, an insect cell, or a mammalian cell. The type of host cell, will, of course, depend on the viral vector employed, and suitable host cell/viral vector combinations will be readily apparent to those of skill in the art.
• In some embodiments, the viral vector is a phage and the host cell is a bacterial cell. In some embodiments, the host cell is anE. colicell. SuitableE. colihost strains will be apparent to those of skill in the art, and include, but are not limited to, New England Biolabs (NEB) Turbo, Top10F′, DH12S, ER2738, ER2267, and XL1-Blue MRF′. These strain names are art recognized and the genotype of these strains has been well characterized. It should be understood that the above strains are exemplary only and that the invention is not limited in this respect.
• In some PACE embodiments, for example, in embodiments employing an M13 selection phage, the host cells areE. colicells expressing the Fertility factor, also commonly referred to as the F factor, sex factor, or F-plasmid. The F-factor is a bacterial DNA sequence that allows a bacterium to produce a sex pilus necessary for conjugation and is essential for the infection ofE. colicells with certain phage, for example, with M13 phage. For example, in some embodiments, the host cells for M13-PACE are of the genotype F′proA⁺B⁺Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlaciZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ.
Kits and Apparatuses
• Some aspects of this disclosure provide kits comprising (a) an expression construct or plasmid as provided herein, wherein the expression construct comprises an inducible promoter controlling at least one of the nucleic acid sequences comprised in the expression construct; and (b) an inducing agent that induces expression from the inducible promoter. In some embodiments, the kit further comprises (c) a vector encoding an M13 phage backbone and a multiple cloning site for insertion of a nucleic acid sequence encoding a gene product to be evolved, wherein the vector or a replication product thereof can be packaged into infectious phage particles in the presence of other phage functions by suitable host cells, but lacks at least one gene required for the generation of infectious particles. In some embodiments, the kit further comprises (d) an accessory plasmid comprising a nucleic acid sequence encoding the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by a desired activity of the gene product to be evolved. In some embodiments, the kit further comprises (e) an accessory plasmid comprising a nucleic acid sequence encoding a dominant-negative version of the at least one gene required for the generation of infectious particles under the control of a promoter that is activated by an undesired activity of the gene product to be evolved. In some embodiments, the kit further comprises a helper phage providing all phage functions except for the at least one gene required for the generation of infectious phage particles provided by the accessory plasmid of (d). In some embodiments, the helper phage or a replication product thereof cannot be packaged into infectious phage particles. In some embodiments, the kit comprises suitable host cells. In some embodiments, the host cells areE. colihost cells.
• Some aspects of this invention provide kits for continuous directed evolution as described herein. In some embodiments, the kit comprises (a) a vector encoding a phage backbone, for example, an M13 phage backbone, and a multiple cloning site for insertion of a nucleic acid sequence encoding a gene of interest. In some embodiments, the vector or a replication product thereof can be packaged into infectious phage particles in the presence of other phage functions by suitable host cells. In some embodiments, the vector or a replication product thereof lacks at least one gene required for the generation of infectious particles.
• In some embodiments, the kit comprises (b) an accessory plasmid comprising a nucleic acid sequence encoding the at least one gene required for the generation of infectious particles under the control of a conditional promoter that is activated by a transcriptional activator.
• In some embodiments, the kit further comprises a helper phage providing all phage functions except for the at least one gene required for the generation of infectious phage particles provided by the accessory plasmid of (b). In some embodiments, the helper phage or a replication product thereof cannot be packaged into infectious phage particles.
• In some embodiments, the kit comprises suitable host cells. In some embodiments, the host cells areE. colihost cells. In some embodiments, the kit further comprises a mutagenesis plasmid. In some embodiments, the mutagenesis plasmid comprising a gene expression cassette encoding umuC (a components ofE. colitranslesion synthesis polymerase V), dam (deoxyadenosine methylase), and/or seqA (a hemimethylated-GATC binding domain), or any combination thereof.
• In some embodiments, a PACE apparatus is provided, comprising a lagoon that is connected to a turbidostat comprising a host cell as described herein. In some embodiments, the host cell is anE. colihost cell. In some embodiments, the host cell comprises a mutagenesis expression construct as described herein, an accessory plasmid as described herein, and optionally, a helper plasmid as described herein, or any combination thereof. In some embodiments, the lagoon further comprises a selection phage as described herein, for example, a selection phage encoding a gene of interest. In some embodiments, the lagoon is connected to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose. In some embodiments, the host cells areE. colicells comprising the F′ plasmid, for example, cells of the genotype F′proA⁺B⁺ Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araDl39 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻.
• For example, in some embodiments, a PACE apparatus is provided, comprising a lagoon of about 100 ml, or about 1 l volume, wherein the lagoon is connected to a turbidostat of about 0.5 l, 1 l, or 3 l volume, and to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose, wherein the lagoon and the turbidostat comprise a suspension culture ofE. colicells at a concentration of about 5×10⁸cells/ml. In some embodiments, the flow of cells through the lagoon is regulated to about 3 lagoon volumes per hour. In some embodiments, cells are removed from the lagoon by continuous pumping, for example, by using a waste needle set at a height of the lagoon vessel that corresponds to a desired volume of fluid (e.g., about 100 ml, in the lagoon. In some embodiments, the host cells areE. colicells comprising the F′ plasmid, for example, cells of the genotype F′proA⁺B⁺Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116)λ⁻.
• Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure.
EXAMPLES
• The potency of some exemplary mutagenesis systems provided herein was characterized, as well as the resulting mutational spectrum. Importantly, this system outperforms commonly used in vivo (XL1-Red, mutA, and LF-Pol I strains) and chemical (EMS, MNNG, and 2AP) methods in mutational efficiency and spectrum, and can even achieve mutational potency and spectrum comparable to that of state-of-the-art in vitro mutagenesis methods (such as PCR mutagenesis with Mutazyme II and Taq polymerase variants). We applied this system to two directed evolution case studies in bacteria and bacteriophage, validating their ability to access biomolecule variants with novel properties in shorter time frames and with greater effectiveness than previously described methods. Collectively, this system substantially advances in vivo mutagenesis capabilities and increases the effectiveness of laboratory evolution efforts.
ResultsMutagenesis Plasmid Minimization
• Bacteria control the rate of chromosomal substitutions through a series of overlapping mechanisms that can be subdivided into three main pathways: proofreading (reduces mutation rate by a factor of ˜10²substitutions/bp/generation), mismatch repair (reduces mutation rate by ˜10³substitutions/bp/generation), and base selection (reduces mutation rate by ˜10⁵substitutions/bp/generation) [10] (FIG. 7). These redundant replication maintenance mechanisms collectively account for the basal substitution rate of bacterial chromosomal DNA of ˜10⁻⁹to 10⁻¹⁰substitutions/bp/generation [10]. Based on prior knowledge of dominant mutators alleles that interfere with DNA replication fidelity, we sought to design a series of small-molecule inducible mutagenesis plasmids (MPs) that offer broad mutational spectra and high levels of mutagenesis in bacterial cells.
• We recently reported the development and application of an MP for in vivo mutagenesis during phage-assisted continuous evolution (PACE) [11]. This MP increases the mutation rate of the M13 bacteriophage ˜100-fold above the basalE. colimutation rate through the arabinose-induced expression of dnaQ926, a dominant negative variant of theE. coliDNA Pol 11H proofreading domain. This plasmid additionally provides umuD′, umuC and recA730, which together allow for in vivo translesion mutagenesis employing UV light or chemical mutagens. This MP (designated MP1) results in a substitution rate of 7.2×10⁻⁵and 5.4×10⁻⁸substitutions/bp/generation for M13 phage andE. coli, respectively [11, 12]. This mutation rate, however, is still several orders of magnitude below the mutation rates provided by conventional in vitro mutagenesis techniques [13].
• Since we sought to avoid the use of exogenous mutagens, we first minimized MP1 by removing umuD′, umuC, and recA730 from MP1 to yield MP2 carrying only dnaQ926, and observed mutation rates in the absence of mutagens to be modestly improved compared to MP1 through a rifampin-resistance assay using the nearly wild-typeE. coliMG1655 ΔrecA::apra (Table 1), enabling an average of 3.6×10⁻⁷substitutions/bp/generation (FIG. 1A). Since dnaQ926 abrogates the proofreading component of DNA replication, we began assessing additional genes that when expressed from the MP can further enhance mutation rate.

• TABLE 1
  Summary of all strains used in this study.
  Strains that were requested from the Yale Coli Genetic
  Stock Center (CGSC) show the corresponding strain numbers.

  Strain	CGSC #	Genotype

  MG1655	12492	F− ΔrecA1918::apra, rph-1 λ−
  ΔrecA
  CSH101	8095	F′ lacI373 lacZ571/ara-600
  		Δ(gpt-lac)5 relA1 spoT1 thiE1 λ−
  CSH102	8096	F′ lacI373 lacZ572/ara-600
  		Δ(gpt-lac)5 relA1 spoT1 thiE1 λ−
  CSH103	8097	F′ lacI373 lacZ573/ara-600
  		Δ(gpt-lac)5 relA1 spoT1 thiE1 λ−
  CSH104	8098	F′ lacI373 lacZ574/ara-600
  		Δ(gpt-lac)5 relA1 spoT1 thiE1 λ−
  CSH105	8099	F′ lacI373 lacZ575/ara-600
  		Δ(gpt-lac)5 relA1 spoT1 thiE1 λ−
  CSH106	8100	F′ lacI373 lacZ576/ara-600
  		Δ(gpt-lac)5 relA1 spoT1 thiE1 λ−
  S1030	N/A	F' proA + B + Δ(lacIZY) zzf.:Tn10
  		lacIQ1PN25-tetR luxCDE/endA1
  		recA1 galE15 galK16 nupG rpsL
  		ΔlacIZYA araD139 Δ(ara, leu)7697
  		mcrA Δ(mrr-hsdRMS-mcrBC)
  		proBA::pir116 araE201 ΔrpoZ
  		Δflu ΔcsgABCDEFG ΔpgaC λ−
  S1021	N/A	F− endA1 recA1 galE15 galK16
  		nupG rpsL ΔlacIZYA araD139
  		Δ(ara, leu)7697 mcrA Δ(mrr-hsdRMS-
  		mcrBC) proBA::pir116 araE201
  		ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ−

DNA Methylation State Manipulation Enhances Mutagenesis
• Mismatch repair reduces the error rate of bacterial DNA replication by a factor of up to 103 [10]. Following daughter strand synthesis, MutSL scan the genome for mismatches that have evaded the base selection and proofreading activities of theE. coliDNA Pol III holoenzyme (FIG. 7). Once recognized, the newly synthesized DNA is nicked by MutH at hemimethylated GATC sequences, then unwound and digested by dedicated helicases and exonucleases. DNA Pol Iii synthesizes a new strand and Dam methylase methylates the resulting DNA. The deletion of mutS, mutL, or mutH, and the overexpression of dam are known to have a strong mutator effect due to impaired mismatch repair [14].
• We added wild-type dam downstream of dnaQ926 on the same arabinose-inducible expression cassette to yield MP3, and observed increased mutagenesis potency but also increased uninduced background mutagenesis, likely due to a strong cryptic σ⁷⁰promoter at the 3′ end of dnaQ926 (FIG. 8). Overall, MP3 resulted in a 3-fold mutagenesis potency increase in the presence of arabinose relative to MP2 but >60-fold increase in background mutagenesis, greatly reducing the dynamic range of induced mutagenesis to only 11-fold (FIG. 1A).
• To restore dynamic range, we installed the gene encoding the hemimethylated GATC-binding domain SeqA downstream of dam. Low-level expression of seqA is known to delay Dam methylation of hemimethylated GATC sequences [15] and induce positive supercoils in chromosomal and episomal DNA [16], which may reduce MP gene transcription in the absence of arabinose. Conversely, high-level seqA expression results in a potent mutator response [17] and induction of negative supercoils in chromosomal and episomal DNA [16], potentially increasing global gene transcription, including that of the MP, upon induction with arabinose. Indeed, the MP carrying dnaQ926, dam, and seqA (MP4) resulted in >60-fold reduced background and 2-fold improved mutagenesis potency in the presence of arabinose relative to MP3, representing a cumulative >100-fold improvement in dynamic range (FIG. 1A).
• While the addition of dam and seqA together increased the mutagenic potency by only 4-fold compared to MP2, it is known that dominant-negative dnaQ mutants partially saturate the mismatch repair response [18], Indeed, including dominant-negative variants of mutS 117, 191, mutL [20, 21], or mutH [21] with dnaQ926 had no additional effect on mutagenesis (Tables 2 and 3). Taken together, these results suggest that the inclusion of seqA and dam on MP4 is sufficient to fully disrupt the mismatch repair response, enabling an average of 4.4×10˜7 substitutions/bp/generation by the rifampicin resistance assay.

• TABLE 2
  Summary of all ORFs carried by the MPs. All MPs use the identical vector
  backbone: a cloDFI3 origin of replication (20-40 copies/cell), a chloramphenicol resistance
  cassette, the arabinose responsive promoter PBADdriving the imitator genes, and the weak
  promoter PCdriving araC. Genes carried by each MP are arranged in the order found in the
  table and are highlighted according to their mechanism of action and/or the canonical repair
  pathway that they disrupt: proofreading (blue), translesion synthesis (purple), methyl-
  directed mismatch repair (red), base excision repair (green), base selection (yellow) and
  unknown (black). Additional optimizations included: codon usage optimization (opt) and
  increased ribosome-binding site strength (*). Boxes are not drawn to scale. Nucleotide and
  protein sequences related to the gene symbols listed in the table above are known to those of
  skill in the art. For gene symbols for which publications are indicated (by superscript
  numbers), any sequences provided in the publication related to the gene symbol is
  incorporated herein by reference. In some embodiments, the gene symbols above relate to
  nucleotide and protein sequences accessible under that gene symbol in any of the National
  Center for Biotechnology Information (NCBI) databases, for example, in the Nucleotide
  Reference Sequence (RefSeq) database, release 69 (Jan. 2, 2015), or in the Unigene
  database available at the time of filing, and any NCBI database entries, e.g., RefSeq database
  entries for the listed genes in the ResSeq database, release 69, or in the Unigene database at
  the time of filing, are incorporated herein by reference.

  Name	Genes encoded on the MP (native order)

  MP1	dnaQ92640	umuD′		umuC		recA73030
  MP2	dnaQ926
  MP-B2	dnaE7442
  MP-B4	dnaE48624
  MP-B5	dnaE102643
  MP-C2	dnaX3645
  MP-C3	dnaX201645
  MP-D3	dnaQ926	dnaE486
  MP-D4	dnaQ926	dnaE1026
  MP-E	dnaQ926	dnaX36
  MP-E2	dnaQ926	dnaX2016
  MP-F2	dnaQ926	mutS53819
  MP-F3	dnaQ926	mutS50319
  MP-H	dnaQ926	mutL705220
  MP-H2	dnaQ926	mutL71320
  MP-H3	dnaQ926	mutL (R261H)21
  MP-H4	dnaQ926	mutL (K307A)21
  MP-I	dnaQ926	mutH (E56A)21
  MP-I2	dnaQ926	mutH (K79E)21
  MP-I3	dnaQ926	mutH (K116E)21
  MP-J	rpsD1247
  MP-J2	rpsD1447
  MP-J3	rpsD1647
  MP3	dnaQ926	dam
  MP-K7	dnaQ926	dam	emrR17
  MP4	dnaQ926	dam	seqA17
  MP-K9	dnaQ926	dam	mutSΔN17
  MP-K10	dnaQ926	dam	seqA	emrR
  MP-K11	dnaQ926	dam	seqA	mutSΔN
  MP-K12	dnaQ926	dam	seqA	dinB42
  MP-K13	dnaQ926	dam	seqA	polB52
  MP-K14	dnaQ926	dam	seqA*
  MP-L	polB
  MP-L2	polB (D156A)52
  MP-P	dnaQ926	dam	seqA	emrR	mutH (ES6A)
  MP-P3	dnaQ926	dam	seqA	emrR	mutL713
  MP-P4	dnaQ926	dam	seqA	emrR	mutS503
  MP-P5	dnaQ926	dam	seqA	emrR	mutSΔN
  MP-P6	dnaQ926	dam	seqA	emrR	dinB
  MP-P7	dnaQ926	dam	seqA	emrR	polB
  MP-P8	dnaQ926	dam	seqA	emrR	Ugi41	AID22
  MP-P9	dnaQ926	dam	seqA	emrR	Ugi	APOBEC122
  MP6	dnaQ926	dam	seqA	emrR	Ugi	CDA122
  MP-P11	dnaQ926	dam	seqA	emrR	Ugi	CDA1	mutSΔN
  MP-Q	dnaQ926	dam	seqA	rsmE44
  MP-Q2	dnaQ926	dam	seqA	cchA44
  MP-Q3	dnaQ926	dam	seqA	yffl41
  MP-Q4	dnaQ926	dam	seqA	yfjY17
  MP-Q5	dnaQ926	dam	seqA	ugi	AID
  MP-Q6	dnaQ926	dam	seqA	ugi	APOBEC1
  MP5	dnaQ926	dam	seqA	ugi	CDA1
  MP-Q8	dnaQ926	dam	seqA	nrdAB25
  MP-Q9	dnaQ926	dam	seqA	nrdA (H59A)B25
  MP-Q10	dnaQ926	dam	seqA	nrdA (A65V)B46
  MP-Q11	dnaQ926	dam	seqA	nrdA (A301V)B46
  MP-Q12	dnaQ926	dam	seqA	nrdAB (P334L)46
  MP-Q13	dnaQ926	dam	seqA	nrdEF25
  MP-R	dnaQ926	dam	seqA	ugi	AID (opt)
  MP-R2	dnaQ926	dam	seqA	ugi	APOBEC1 (opt)
  MP-R3	dnaQ926	dam	seqA	ugi	CDA1 (opt)
  MP-R4	dnaQ926	dam	seqA	emrR	ugi	AID (opt)
  MP-R5	dnaQ926	dam	seqA	emrR	ugi	APOBEC1 (opt)
  MP-R6	dnaQ926	dam	seqA	emrR	ugi	CDA1 (opt)
  MP-S	dnaQ926	MAG149

  MP-S2	dnaQ926	AAG (Y127I-H136L)50
  MP-S3	dnaQ926	Δ80-AAG (Y127I-H136L)50

  MP-T	dnaQ926	dam	seqA	emrR	ugi	AID (7)51
  MP-T2	dnaQ926	dam	seqA	emrR	ugi	AID (73)51
  MP-T3	dnaQ926	dam	seqA	emrR	ugi	AID (7.33)51
  MP-T4	dnaQ926	dam	seqA	emrR	ugi	AID (7.3.3)51
  MP-T5	dnaQ926	dam	seqA	emrR	ugi	AID (7.3.1)51
  MP-T6	dnaQ926	dam	seqA	emrR	ugi	AID (7.3.2)51
  MP-U	dnaQ926*	dam	seqA	emrR	ugi	CDA1
  MP-U2	dnaQ926	dam*	seqA	emrR	ugi	CDA1
  MP-U3	dnaQ926	dam	seqA	emrR*	ugi	CDA1
  MP-U4	dnaQ926	dam	seqA	emrR	ugi	CDA1*
  MP-V	BR1353	dam	seqA	emrR	ugi	CDA1
  MP-V2	BRM153	dam	seqA	emrR	ugi	CDA1
  MP-V3	BR1153	dam	seqA	emrR	ugi	CDA1
  MP-V4	BR653	dam	seqA	emrR	ugi	CDA1
  MP-V5	BR153	dam	seqA	emrR	ugi	CDA1

• TABLE 3
  Summary of induced and uninduced mutagenesis levels for all
  designed MPs. All MPs were tested using the rifampin resistance
  assay to assess their relative mutagenic load under uninduced
  (glucose) and induced (arabinose) conditions. The viability of the
  MP-carrying strains under the induced conditions (as a percentage
  of the viability of the strain without an MP) is also shown.
  Ideal MPs show low background and high induced
  mutagenesis, with only moderate reductions in viability.

  Name	Uninduced μbp	Induced μbp	Viability (%)

  None	1.20E−10	2.20E−11	100
  MP1	3.00E−09	2.40E−07	87.5
  MP2	1.00E−09	3.60E−07	136.8
  MP-B2	0.00E+00	0.00E+00	25.4
  MP-B4	0.00E+00	0.00E+00	45.8
  MP-B5	0.00E+00	0.00E+00	55.9
  MP-C2	0.00E+00	0.00E+00	63.6
  MP-C3	0.00E+00	0.00E+00	53.4
  MP-D3	0.00E+00	8.50E−09	24.2
  MP-D4	0.00E+00	9.50E−09	68.6
  MP-E	0.00E+00	7.80E−10	61
  MP-E2	1.50E−09	2.50E−09	40.7
  MP-F2	3.60E−09	9.80E−09	48.3
  MP-F3	0.00E+00	1.70E−08	73.7
  MP-H	0.00E+00	2.60E−08	94.1
  MP-H2	2.60E−09	3.50E−08	35.6
  MP-H3	4.70E−10	1.00E−08	71.2
  MP-H4	0.00E+00	7.40E−09	63.6
  MP-I	0.00E+00	1.60E−08	61
  MP-I2	0.00E+00	6.40E−09	83.9
  MP-I3	0.00E+00	1.00E−08	89
  MP-J	0.00E+00	0.00E+00	223.7
  MP-J2	0.00E+00	0.00E+00	142.4
  MP-J3	0.00E+00	0.00E+00	137.3
  MP3	6.40E−08	9.70E−07	31.2
  MP-K7	2.40E−09	3.30E−07	9.4
  MP4	1.00E−09	1.60E−06	22.9
  MP-K9	5.70E−09	3.30E−07	23.4
  MP-K10	4.10E−11	1.10E−07	80.6
  MP-K11	3.60E−10	6.80E−08	63.6
  MP-K12	9.80E−10	5.40E−08	86.4
  MP-K13	0.00E+00	6.70E−08	63.6
  MP-K14	4.10E−09	0.00E+00	0.8
  MP-L	0.00E+00	0.00E+00	129.7
  MP-L2	0.00E+00	0.00E+00	93.8
  MP-P	1.90E−09	1.40E−08	144.9
  MP-P3	4.20E−09	9.20E−07	33.3
  MP-P4	1.70E−08	5.70E−07	81.4
  MP-P5	1.00E−09	2.20E−06	26.7
  MP-P6	1.80E−09	4.20E−07	10.7
  MP-P7	1.30E−09	2.80E−07	83.9
  MP-P8	2.00E−09	4.80E−07	32.8
  MP-P9	1.20E−07	2.60E−05	2.9
  MP6	9.00E−09	2.30E−05	1.7
  MP-P11	6.10E−09	1.90E−05	1.4
  MP-Q	1.60E−09	4.90E−07	14.7
  MP-Q2	5.70E−09	3.40E−07	20.6
  MP-Q3	8.70E−10	3.40E−07	16.8
  MP-Q4	7.10E−09	9.70E−07	15
  MP-Q5	7.00E−09	4.00E−07	22.5
  MP-Q6	3.60E−09	6.70E−07	16.3
  MP5	1.80E−08	7.40E−06	3.8
  MP-Q8	6.20E−10	1.80E−07	37.4
  MP-Q9	9.20E−10	6.90E−08	73.7
  MP-Q10	8.30E−09	3.80E−08	81.4
  MP-Q11	4.30E−10	4.20E−08	66.1
  MP-Q12	8.80E−10	2.00E−08	144.9
  MP-Q13	1.50E−09	1.60E−08	101.7
  MP-R	3.50E−09	6.00E−07	13.9
  MP-R2	2.80E−08	8.70E−07	13.1
  MP-R3	4.10E−08	4.30E−06	4.3
  MP-R4	8.60E−09	2.50E−06	3.7
  MP-R5	1.80E−07	1.60E−05	5.8
  MP-R6	2.10E−08	6.30E−05	0.7
  MP-S	1.20E−10	2.30E−08	115.3
  MP-S2	6.30E−11	2.10E−08	313
  MP-S3	2.50E−10	9.80E−08	428.4
  MP-T	7.00E−10	1.00E−06	13.6
  MP-T2	7.10E−09	3.50E−07	23.1
  MP-T3	3.20E−08	5.70E−07	12.6
  MP-T4	2.20E−08	8.90E−07	13.7
  MP-T5	2.30E−08	6.00E−07	21.7
  MP-T6	9.20E−09	8.40E−07	16
  MP-U	7.00E−09	9.80E−05	0.6
  MP-U2	2.90E−09	1.10E−05	1.7
  MP-U3	2.40E−08	5.50E−06	2.8
  MP-U4	5.00E−09	9.20E−06	3.7
  MP-V	1.70E−09	5.40E−07	5.7
  MP-V2	2.90E−09	1.10E−06	7.9
  MP-V3	1.10E−08	2.40E−06	3.4
  MP-V4	5.00E−09	2.20E−06	4.6
  MP-V5	8.70E−10	1.90E−06	6.2

Cytosine Deamination and Reduced Base Excision Repair
• Overexpression of the catalytic domains of several cytidine deaminases inE. colihas been shown to have a mutagenic effect, resulting in primarily C→T transitions through a deoxyuracil intermediate [22]. Thecytidine deaminase CDA 1 fromPetromyzon marinusis reported to mediate the efficient mutation of prokaryotic and eukaryotic genomic DNA [22]. Additional mutations or deletion of theE. coliuracil-DNA glycosylase ung synergize with the effect of the deaminase and can enhance mutagenesis by disruption of the native uracil-excision repair pathway [22] (FIG. 7). Two natural protein inhibitors of Ung, Ugi and p56 [23], inhibit Ung through mimicry of structural and electronic features of uracil-containing DNA [23]. We hypothesized that the inclusion of a cytidine deaminase and uracil-DNA glycosylase inhibitor would further increase the potency of the MP through impairment of the uracil-excision repair pathway.
• We placed ugi and cda1 downstream of dnaQ926, dam and seqA to yield MP5, and observed a 5-fold mutagenesis potency increase under induced conditions, representing an 18-fold increase in background mutagenesis compared to MP4 (FIG. 1A). This background increase was caused by two predicted σ⁷⁰promoters at the 3′ end of the seqA open reading frame, resulting in constitutive ugi and cda1 transcription (FIG. 9). We considered this background mutation rate for MP5 to be acceptable, as it was only ˜5-fold higher than the starting MP1. Alternative cytosine deaminases, including rat APOBEC1 and human AID, generally resulted in weaker effects on mutation rate than CDA1, in agreement with previous reports (CDA1>>AID≈APOBEC1) [22] (Tables 2 and 3). Overall, MP5 yielded 2.0×10⁻⁶substitutions per bp per generation, a 31-fold increase in mutation rate relative to MP1 (Tables 2 and 3).
Impairing Mutagenic Nucleobase Export
• Two major determinants of base selection during DNA replication are the catalytic alpha subunit ofE. coliDNA Pol III, and regulation of the intracellular pools of dNTPs available during replication (FIG. 7). Mutations affecting the former are generally not viable or exert a mutator effect through reduced affinity to the proofreading domain, DnaQ [24], whereas perturbations affecting the latter are generally more tolerated and can be modified to affect the mutational spectrum [25]. We screened a number of mutator proteins that are known to compromise intracellular dNTP pools, and found overexpression of the emrR transcriptional repressor to be the most promising (FIG. 1A; Tables 2 and 3).

• TABLE 4
  Comparison of MP1-MP6 with previously described mutator plasmids. In each case, the mutator genes are listed with the source
  organism(s): Ee =Escherichiacoli; Sc = Saccharomyces cerevisiae; Hs = Homo sapiens; Rn = Rattus norvegicus; Pa =Pseudomonas
  aeruginosa; Ll =Lactococcuslactis; PBS2 =Bacillussubtilis phage PBS2; Pm = Petromyzon marinus. In all cases, the fraction of
  cells showing rifampin resistance (RifR) was used to calculate μbpas described in the methods section, using R = 21 sites,
  N = 108, and N0= 1.5 × 107to approximate the levels as compared to the MP1-6 series. The fold increase in mutagenesis is shown for
  each MP (defined as the ratio of the mutagenesis in the strain without the MP vs. in the strain with the MP).
  All MPs are compared to MP6 in total mutagenesis efficiancy. *In cases where the MP was inducible, the dynamic range represents
  the fold increase between the uninduced and induced states for strains carrying the MP.

  Source

  Fraction RifR

  μbp(bp−1 generation−1)

  vs MP6

  Dynamic

  organism	Gene(s)	−MP	+MP	−MP	+MP	Fold	(%)	range*	Ref.

  Ec	Dam	8.10E+08	3.00E−07	2.035+07	7.53E−09	0	0	—	48
  Ec	dnaE173	5.00E−09	7.20E−06	1.26E−10	1.81E−07	1440	1	24	24
  Ec	dnaQ926	3.00E−08	3.47E−04	7.53E−10	8.71E−06	11567	38	—	40
  Ec	mutD5	3.00E−08	5.70E−05	7.53E−10	1.43E−06	1900	6	—	40
  Sc	mag1	5.13E−08	3.61E−06	1.29E−09	9.06E−08	70	0	—	49
  Sc	mag1	1.00E−08	2.00E−06	2.51E−10	5.02E−08	200	0	200	54
  Ec	dinB	4.00E−08	4.55E−06	1.00E−09	1.14E−07	114	0	114	55
  Ec	dnaE (K655Y)	1.00E−08	8.45E−06	2.51E−10	2.12E−07	845	1	—	42
  Hs	AID	1.30E−08	1.03E−07	3.26E−10	2.59E−09	8	0	8	56
  Rn	APOBEC1	2.53E−08	1.23E−05	6.36E−10	3.09E−07	486	1	14	57
  Rn	APOBEC2	2.53E−08	2.50E−08	6.36E−10	6.28E−10	1	0	—	57
  Hs	AID	2.53E−08	1.66E−07	6.36E−10	4.17E−09	7	0	—	57
  Hs	APOBEC3C	2.53E−08	2.93E−07	6.36E−10	7.35E−09	12	0	—	57
  Hs	APOBEC3G	2.53E−08	2.70E−07	6.36E−10	6.78E−09	11	0	—	57
  Ec	mutH (E56A)	4.40E−08	8.74E−06	1.10E−09	2.19E−07	199	1	—	21
  Ec	mutH (K116E)	4.40E−08	8.40E−06	1.10E−09	2.11E−07	191	1	—	21
  Ec	mutH (K79E)	4.40E−08	7.00E−06	1.10E−09	1.76E−07	159	1	—	21
  Ec	mutH (B77A)	4.40E−08	6.94E−06	1.10E−09	1.74E−07	158	1	—	21
  Ec	mutH (D70A)	4.40E−08	6.88E−06	1.10E−09	1.73E−07	156	1	—	21
  Ec	mutH CΔ5	4.40E−08	9.10E−07	1.10E−09	2.28E−08	21	0	—	21
  Ec	mutL (R95F/N302A)	4.40E−08	1.17E−05	1.10E−09	2.94E−07	267	1	—	21
  Ec	mutL (R261H)	4.40E−08	1.04E−05	1.10E−09	2.62E−07	237	1	—	21
  Ec	mutL (E29A)	4.40E−08	8.32E−06	1.10E−09	2.09E−07	189	1	—	21
  Ec	mutL (K307A)	4.40E−08	6.75E−06	1.10E−09	1.69E−07	153	1	—	21
  Ec	mutL (N302A)	4.40E−08	3.50E−06	1.10E−09	8.79E−08	80	0	—	21
  Ec	mutL (R95F)	4.40E−08	1.27E−07	1.10E−09	3.19E−09	3	0	—	21
  Ec	mutL (K159E)	4.40E−08	7.26E−06	1.10E−09	1.82E−07	165	1	—	21
  Ec	mutL (R266E)	4.40E−08	6.05E−06	1.10E−09	1.52E−07	138	1	—	21
  Ec	mutL (R177E)	4.40E−08	1.77E−06	1.10E−09	4.44E−08	40	0	—	21
  Ec	mutL (I90R)	4.40E−08	1.56E−07	1.10E−09	3.92E−09	4	0	—	21
  Ec	mutL (R237E)	4.40E−08	1.40E−07	1.10E−09	3.51E−09	3	0	—	21
  Ec	mutL (G238A)	4.40E−08	5.40E−08	1.10E−09	1.36E−09	1	0	—	21
  Ec	mutL (G238D)	4.40E−08	1.03E−05	1.10E−09	2.59E−07	234	1	—	21
  Ec	mutL (I90E)	4.40E−08	1.70E−08	1.10E−09	4.27E−10	0	0	—	21
  Ec	mutS (S668A/T669V)	4.40E−08	1.33E−05	1.10E−09	3.34E−07	303	1	—	21
  Ec	mutS (K620M)	4.40E−08	1.21E−05	1.10E−09	3.05E−07	276	1	—	21
  Ec	mutS (D693A)	4.40E−08	1.15E−05	1.10E−09	2.90E−07	262	1	—	21
  Ec	mutS (E694Q)	4.40E−08	1.13E−05	1.10E−09	2.84E−07	257	1	—	21
  Ec	mutS (E694A)	4.40E−08	8.84E−06	1.10E−09	2.22E−07	201	1	—	21
  Ec	mutS (E694A/S668A/T669V)	4.40E−08	8.02E−06	1.10E−09	2.01E−07	182	1	—	21
  Ec	mutS (D693N)	4.40E−08	5.58E−06	1.10E−09	1.40E−07	127	1	—	21
  Ec	mutS (H760A)	4.40E−08	8.01E−07	1.10E−09	2.01E−08	18	0	—	21
  Ec	mutS (H728A)	4.40E−08	4.48E−07	1.10E−09	1.12E−08	10	0	—	21
  Ec	mutS (F596A)	4.40E−08	3.14E−07	1.10E−09	7.88E−09	7	0	—	21
  Ec	mutS (S612A)	4.40E−08	3.70E−08	1.10E−09	9.29E−10	1	0	—	21
  Ec	mutS (F36A)	4.40E−08	1.06E−05	1.10E−09	2.66E−07	241	1	—	21
  Ec	mutS (E38Q)	4.40E−08	4.19E−06	1.10E−09	1.05E−07	95	0	—	21
  Ec	mutS (D162R/E164R)	4.40E−08	4.30E−06	1.10E−09	1.08E−07	98	0	—	21
  Ec	mutS (R163E)	4.40E−08	3.90E−08	1.10E−09	9.79E−10	1	0	—	21
  Ec	mutS (R197E/R193E)	4.40E−08	1.11E−05	1.10E−09	2.79E−07	253	1	—	21
  Ec	mutS (R197E/R198E/R199E)	4.40E−08	6.15E−06	1.10E−09	1.54E−07	140	1	—	21
  Ec	mutS (E177A)	4.40E−08	1.10E−05	1.10E−09	2.75E−07	249	1	—	21
  Ec	mutS (T115A)	4.40E−08	7.99E−06	1.10E−09	2.01E−07	132	1	—	21
  Ec	Dam	3.00E−08	2.00E−06	7.53E−10	5.02E−08	67	0	—	17
  Ec	emrR	3.00E−08	5.00E−06	7.53E−10	1.26E−07	167	1	—	17
  Ec	mutS*	3.00E−08	3.00E−07	7.53E−10	7.53E−09	10	0	—	17
  Ec	seqA	3.00E−08	8.00E−07	7.53E−10	2.01E−08	27	0	—	17
  Ec	dinB	3.00E−08	3.00E−07	7.53E−10	7.53E−09	10	0	—	17
  Pa	nfx3	8.00E−08	3.50E−06	2.01E−08	8.79E−08	44	0	—	26
  Ll	dnaN	8.00E−08	9.80E−08	2.01E−09	2.46E−09	1	0	—	26
  Ll	dnaA	8.00E−08	1.90E−07	2.01E−09	4.77E−09	2	0	—	26
  Ll	uvrA, ysjE	8.00E−08	3.90E−08	2.01E−09	9.79E−10	0	0	—	26
  Ll	uvrA	8.00E−08	1.10E−07	2.01E−09	2.76E−09	1	0	—	26
  Ll	mhA, sipL, purR	8.00E−08	4.80E−08	2.01E−09	1.20E−09	1	0	—	26
  Ll	mhA, sipL	8.00E−08	6.70E−08	2.01E−09	1.68E−09	1	0	—	26
  Ll	nhA	8.00E−08	1.40E−08	2.01E−09	3.51E−10	0	0	—	26
  Ec	polB	2.20E−08	2.32E−06	5.52E−10	5.82E−08	105	0	116	52
  Ec	polB Q779V	2.20E−08	6.14E−08	5.52E−10	1.54E−09	3	0	—	52
  Ec	polB Δ780-783	2.20E−08	5.93E−08	5.52E−10	1.49E−09	3	0	—	52
  Ec	polBB (D156A)	2.20E−08	4.73E−04	5.52E−10	1.19E−05	21478	52	—	52
  Ec	uvrAB	7.90E−09	5.40E−08	1.98E−10	1.36E−09	7	0	—	58
  Ec	uvrABC	7.90E−09	1.90E−07	1.98E−10	4.77E−09	24	0	—	58
  Hs	AID	1.00E−08	1.40E−07	2.51E−10	3.51E−09	14	0	—	51
  Hs	AID (K10E/E156G)	1.00E−08	6.30E−07	2.51E−10	1.58E−08	63	0	—	51
  Hs	AID (K34E/K160E)	1.00E−08	4.20E−07	2.51E−10	1.05E−08	42	0	—	51
  Hs	AID	2.40E−08	4.90E−07	6.02E−10	1.23E−08	20	0	—	59
  Hs	AID-3FL	2.40E−08	3.70E−07	6.02E−10	9.29E−09	15	0	—	59
  Hs	AID-3GL	2.40E−08	1.60E−07	6.02E−10	4.02E−09	7	0	—	59
  Hs	AAG	1.00E−09	2.00E−07	2.51E−11	5.02E−09	200	0	1	50
  Hs	AAG (Y127I/H136L)	1.00E−09	1.15E−05	2.51E−11	2.89E−07	11500	1	4	50
  Ec	nrdAB	1.00E−08	8.00E−08	2.51E−10	2.01E−09	8	0	—	25
  Ec	nrdEF	1.00E−08	3.50E−07	2.51E−10	8.79E−09	35	0	—	25
  Ec	nrdA (H59A)B	1.00E−08	3.80E−07	2.51E−10	9.54E−09	38	0	—	25
  Hs	APOBEC3G	2.60E−07	2.40E−06	6.53E−09	6.02E−08	9	0	—	60
  Hs	APOBEC3G (E259Q)	2.60E−07	3.60E−07	6.53E−09	9.04E−09	1	0	—	60
  Hs	APOBEC3G (E254R)	2.60E−07	3.50E−06	6.53E−09	8.79E−08	13	0	—	60
  Hs	APOBEC3G (R313R)	2.60E−07	2.60E−07	6.53E−09	6.53E−09	1	0	—	60
  Hs	APOBEC3G (R320E)	2.60E−07	5.80E−07	6.53E−09	1.46E−08	2	0	—	60
  Hs	APOBEC3G (R313E/R320E)	2.60E−07	3.10E−07	6.53E−09	7.78E−09	1	0	—	60
  Hs	APOBEC3G (R374E)	2.60E−07	5.50E−07	6.53E−09	1.38E−08	2	0	—	60
  Hs	APOBEC3G (R376E)	2.60E−07	1.90E−06	6.53E−09	4.77E−08	7	0	—	60
  Hs	APOBEC3G (R374E/R376E)	2.60E−07	6.90E−07	6.53E−09	1.73E−08	3	0	—	60
  Hs	APOBEC3G (R213E)	2.60E−07	4.40E−07	6.53E−09	1.10E−08	2	0	—	60
  Hs	APOBEC3G (R215E)	2.60E−07	3.20E−07	6.53E−09	8.03E−09	1	0	—	60
  Hs	APOSEC3G (R213E/215E)	2.60E−07	2.90E−07	6.53E−09	7.28E−09	1	0	—	60
  Ec	dnaQ926, umuD′, umuC, recA730	7.35E−09	2.15E−05	7.10E−11	2.36E−07	3320	1	78	12
  Ec	dnaQ926	7.35E−09	2.98E−05	7.10E−11	3.61E−07	5087	2	355	This work
  Ec	dnaQ926, dam	7.35E−09	5.54E−05	7.10E−11	9.74E−07	13723	4	15	This work
  Ec	dnaQ926, dam, seqA	7.35E−09	1.03E−04	7.10E−11	1.60E−06	22610	7	1604	This work
  Ec, PBS2,	dnaQ926, dam, seqA, ugi, cda1	7.35E−09	2.88E−04	7.10E−11	7.36E−06	103749	32	419	This work
  Pm
  Ec, PBS2,	dnaQ926, dam, seqA, emrR, ugi, cda1	7.35E−09	4.73E−04	7.10E−11	2.29E−05	322414	100	34941	This work
  Pm

• Derepression by EmrR results in upregulation of emrAB, which produces a multidrug efflux pump responsible for antibiotic resistance and the putative export of mutagenic nucleobase intermediates [26]. In an unbiased screen, emrR overexpression was found to have a potent mutator effect [17], presumably as a consequence of retaining these mutagenic nucleobases within the cell. To decrease background mutagenesis compared to MP5, we placed the emrR cassette between seqA and ugi, thereby disrupting the strong predicted σ⁷⁰promoter, yielding MP6. MP6 exhibited 2-fold lower background mutagenesis, while improving the overall mutator effect under induced conditions by 3-fold (FIG. 1A).
• Features of the MP6 Mutagenesis System and Comparison with Other Methods
• We chose MP6 for in-depth characterization because it offered the highest mutagenic potency with acceptable levels of toxicity (>1% of cells surviving under maximal MP induction) under the tested conditions. Efforts to increase the expression level of the six key genes in MP6 resulted in substantially higher toxicity or reduced potency (Tables 2 and 3). When induced, MP6 results in a 322.000-fold increase in mutation rate of chromosomal DNA over that of wild-typeE. coli, and a 100-fold increase in mutation rate relative to that of MP1. Induced MP6 results in an average of 2.3×10⁻⁵substitutions/bp/generation, representing to our knowledge the most potent inducible and genetically encodable mutagenesis method in bacteria reported to date (Table 4). This potency compares favorably to overexpression of PolB (D156A) (μ_bp=3.2×10⁻⁶), dnaQ926 (μ_bp=2.4×10⁻⁶), or mutD5 (μ_bp=3.9×10⁻⁷) (Table 4). We note that most previously published mutagenic constructs suffer from a lack of inducibility, have deleterious effects on host viability, and require overexpression of the mutagenic protein. In contrast, the MPs described here rely on the low-level expression of multiple genes, thereby affecting multiple pathways and enabling broad mutagenic spectra.
• Additional permutations of this design or the inclusion of alternative mutators impaired overall mutation rate, or strongly decreased host viability, as evidenced by the characteristics of 80 candidate MPs with mutation rates spanning five orders of magnitude (FIGS. 10, 11, and 12; Tables 2 and 3). We observed that a loss of bacterial viability proportional to mutagenic potency beyond a mutation rate of ˜4×10⁻⁷substitutions per bp per generation, corresponding to an average of ˜1.9 substitutions/genome/generation. Given that −10-30% of theE. coligenome has been estimated to be essential [27], this mutation rate threshold corresponds to ˜0.2-0.6 mutation in an essential gene/generation.
• Next we tested the dynamic range and inducibility of MP6. To limit background mutagenesis, we increased the concentration of glucose from 25 mM to 200 mM during the transformation and growth stages prior to induction to maximize catabolite repression of the arabinose-inducible promoter. Under these modified conditions, induction of log-phase cultures of MG1655 ΔrecA::apra carrying MP6 with increasing concentrations of arabinose revealed a 35,000-fold dynamic range between 10 μM and 100 mM arabinose (FIG. 2A). Despite this very strong induction effect, MP6-carrying cultures maintained low levels of mutagenesis when suppressed with 200 mM glucose (FIG. 2A).
• To further evaluate MP6, we compared its performance with that of the most commonly used in vivo mutagenesis strain, XL1-Red (Agilent Technologies). XL1-Red is deficient in proofreading (mutD5), mismatch-repair (mutS) and base-excision (mutT) activities, resulting in high levels of substitutions in chromosomal and episomal DNA [6]. However, the strain grows very slowly, is difficult to manipulate genetically, has poor transformation efficiency, and produces a fairly narrow mutagenic spectrum [7]. Using the rifampin resistance assay, we found that XL1-Red results in 29 substitutions/genome/generation, while MP6 in the induced state produces an average of 110 substitutions/genome/generation, a ˜4-fold higher mutation rate (FIGS. 2B, 2C). The uninduced state of MP6 yields similar background mutagenesis levels (0.01 substitutions/genome/generation) as the non-mutagenic related strain XL1-Blue (0.005 substitutions/genome/generation) (FIGS. 1A, 1B, 2C). Together, these results establish that MP6 allows for mutagenesis levels exceeding that of the most commonly used mutatorE. colistrain, and offers the ability to control mutation rate with an exogenous small molecule.
MP6 Augments M13 Bacteriophage Mutagenesis
• To further characterize MP1, MP4, and MP6, we assessed their impact on the mutagenesis of bacteriophage DNA. Phage display is a widely used platform for laboratory screening and evolution efforts, and has been used to generate many proteins with novel or improved activities [28]. We measured the mutagenesis rate of M13 phage in host cells harboring a variety of MP variants or in XL1-Red using a lacZ inactivation assay. Briefly, a lacZ cassette was integrated downstream of geneIII in the wild-type M13 genome to yield SP063, resulting in high-level expression of β-galactosidase and the production of blue plaques in the presence of an X-Gal analog (FIG. 13). Disruption of the lacZ cassette due to high mutagenesis reduces or ablates this blue-plaque phenotype, enabling the estimation of phage mutagenesis rates. We compared the ratio of white:blue plaques (lacZ phenotype) using MP1, MP4, MP6, and XL1-Red.
• Under conditions supporting phage inoculum expansion in overnight culture using S1030 cells (FIG. 14 and Table 1), we observed that the phage-borne lacZ inactivation rates scaled with MP potency, reaching up to 27% white plaques (representing mutant, inactive lacZ genes) after 18 to 24 h of culture with MP6 (FIG. 3A). As MP6 increases the rate of mutagenesis by ˜100-fold as compared to MP1, the expected M13 bacteriophage mutation rate is elevated to 7.2×10⁻³substitutions/bp/generation [11], resulting in an average of 22 substitutions/genome/generation in the SP063 phage. This mutagenesis potency of 2.3 substitutions/kb (0.23%) approaches the potency of the most commonly used in vitro mutagenesis methods, Mutazyme II (0.3%-1.6%, Agilent Technologies).
• To allow for a comparison to XL1-Red, which lacks an F′ episome and thus cannot be infected by M13 phage particles but allows for the production of fully functional phage, purified SP063 dsDNA was transformed into S1021 (the F⁻ variant of S1030, Table 1) cells carrying the aforementioned MPs (induced prior to or after SP063 transformation, or both;FIG. 15) or XL1-Red. Whereas phage produced from transformed XL1-Red cells only yielded an average of 5% white plaques on S1030 cells (FIG. 3B), phage produced from the S1021 strain carrying MP6 yielded 15% white plaques and MP1 and MP4 yielded 7% and 10% white plaques, respectively, on S1030 cells (FIG. 3C). These results further demonstrate the greater mutational potency of MP6 compared to that of XL1-Red, and also highlight the strain flexibility achieved by using inducible, genetically encodable mutagenesis systems.
Mutational Spectra of Designed MPs
• In addition to mutagenic potency, the distribution of mutation types is also important, as a narrow mutational spectrum limits the diversity of changes that can be accessed. To analyze the spectrum of produced mutations, we took advantage of previously reported distributions of rifampin-resistant rpoB mutants. Importantly, mutations covering each of the 12 possible transitions and transversions in the rpoB gene are known to endowE. coliwith resistance to high levels of rifampin [29]. We assessed the spectra of MP1, MP4, and MP6 by sequencing rifampin-resistant rpoB alleles in mutated MG1655 ΔrecA::apra, and compared the spectra of each to the rpoB mutation spectrum afforded by XL1-Red (FIGS. 4A-D). MP1 yielded a narrow mutagenic spectrum strongly biased towards A:T→T:A transversions, a known side-effect of using recA730-based mutators on genomic DNA [30]. Comparatively, the intermediate MP4 had a more uniform distribution, covering more transitions and a marked increase in G:C→A:T and A:T→G:C, a hallmark of mutagenesis methods that perturb the mismatch repair response [31]. MP6 exhibited a wider spectrum still, with a more uniform distribution of transitions and transversions. The identities of the observed rifampin-resistant mutations are in agreement with previous studies (FIG. 4E) [29]. Notably, XL1-Red exclusively displayed two types of mutations, C:G→T:A and T:A→C:G transitions (FIGS. 4D, 4E). This observation is consistent with previous reports describing the narrow mutational spectrum of XL1-Red [7]. Additional MP characterization using the established β-galactosidase (lacZ) reversion strains developed by Miller and coworkers [32] revealed similar mutational spectra as observed in the rifampin resistance assays (FIG. 16).
• To further characterize the mutagenic spectra, we propagated the lacZ-encoding phage SP063 using 51030 strains carrying MP1, MP4 and MP6, or transformed SP063 DNA directly into XL1-Red to produce progeny phage, and subjected the lacZ ORF from all the resultant phage to high-throughput DNA sequencing (FIGS. 4F-I). The mutagenesis efficiency for all conditions was in agreement with that revealed by the other assays (FIGS. 4A-I,16). The MPs generated broad mutagenic spectra in progeny phage, consistent with both rpoB single clone sequencing and lacZ reversion assays, with the exception of MP1, which showed a more uniform distribution of mutation types using the phage assay and lacZ reversion assays than using rpoB sequencing. This discrepancy is likely a result of the MP1-encoded recA730 allele, which selectively enhances the rate of A:T→T:A transversions in DNA of strains lacking recA (MG1655 ΔrecA::apra), as it no longer competes for substrates with wild-type (CSH strains [32], Table 1) or reduced-activity recA mutants (S1021 and S1030, Table 1) for function. Phage sequences produced from XL1-Red revealed a much narrower mutational spectrum, with a bias for A:T→C:G mutations. Taken together, these results reveal that the MPs developed in this study can outperform the most widely used in vivo and in vitro mutagenesis techniques (FIG. 17) both in mutagenic potency and mutational breadth.
Evolution of Antibiotic Resistance Using the Designed MPs
• To evaluate the impact of these MPs on laboratory evolutionary outcomes, we evolved antibiotic resistance inE. coli, as well as RNA polymerase substrate specificity changes in bacteriophage, using cells harboring different MPs. First we tested the ability of MP1, MP4, or MP6 to evolve resistance ofE. colistrain MG1655 ΔrecA::apra to a number of commonly used antibiotics. Mid-log-phase cultures of MG1655 ΔrecA::apra carrying MP1, MP4, or MP6 were induced for 18-21 h, then serially diluted and plated on agar plates without antibiotics, or with 5-100 μg/mL of carbenicillin, cephotaxime, fosfomycin, kanamycin, metronidazole, norfloxacin, rifampin, spectinomycin, streptomycin, or tetracycline. The antibiotic concentrations used in all ten cases were well above known MIC values (Table 5).

• TABLE 5
  Minimum inhibitory concentrations (MICs) for selected antibiotics.
  All data regardingE. coli MICs was tabulated from the
  Antimicrobial Index Knowledgebase (antibiotics.toku-e.com).

  	Antibiotic	MIC (ug/mL)
  	carbenicillin	2-25
  	cefotaxime	0.016-0.25
  	fosfomycin	0.125-8
  	kanamycin	0.25-8
  	metronidazole	8-32
  	norfloxacin	0.016-0.125
  	rifampin	0.5-16
  	spectinomycin	8-64
  	streptomycin	1-16
  	tetracycline	0.5-8

• After only 18 hours of growth on antibiotic-containing solid medium, large fractions of the bacterial population showed resistance to high concentrations of carbenicillin, fosfomycin, kanamycin, metronidazole, rifampin, spectinomycin, streptomycin, and tetracycline (FIG. 5A and Table 6). No resistant colonies were detected for cefotaxime (a third-generation cephalosporin) or norfloxacin (a synthetic fluoroquinolone). The frequency of antibiotic resistance strongly correlated with MP potency (FIG. 5A). For example, we observed the evolution of resistance to high levels of kanamycin with no intermediate selection step using MP6, which allowed for up to 11% of the total population to grow in the presence of 30 μg/mL kanamycin. In contrast, only 0.02% or 0.66% of the population survived this level of kanamycin when using MP1 or MP4, respectively (FIG. 5A).

• TABLE 6
  Comparison of developed MPs to chemical mutagens, UV light and XL1-Red.
  The fraction of cells resistant to each antibiotic upon mutagenic treatment is shown for all
  tested antibiotics. No resistance was observed for norfloxacin using any MP, chemical
  mutagen, or strain. All MPs, chemical mutagens, and UV light treatments usedE.coli
  MG1655 ΔrecA::apra. CRB, 50 μg/mL carbenicillin; CTX, 5μg/mL cefotaxime; FOS, 100 μg/mL
  fosfomycin; KAN, 30 μg/mL kanamycin, MTX, 100 μg/mL metronidazole; RIF, 100 μg/mL
  rifampin; SPC, 100 μg/mL spectinomycin; STR, 50 μg/mL streptomycin; TET, 10
  μg/mL tetracycline. We note that MP1 (recA730), XL1-Blue (recA1), and XL1-Red (wt recA)
  are all proficient at recombination, a known requirement for high-level resistance to
  metronidazole61. Additionally, XL1-Blue and XL1-Red are both inherently resistant to
  tetracycline, explaining the observed high incidence of resistance.

  	CRB	CTX	FOS	KAN	MIX	RIF	SPC	STR	TET
  no MP	0.00E+00	0.00E+00	1.10E−05	5.78E−06	2.22E−07	5.56E−08	0.00E+00	6.67E−07	0.00E+00
  MP1	9.57E−06	0.00E+00	9.17E−04	1.84E−04	6.47E−01	1.26E−05	1.91E−07	5.68E−06	0.00E+00
  MP4	1.37E−05	0.00E+00	6.97E−03	6.56E−03	1.52E−06	6.17E−05	1.14E−05	1.56E−05	5.97E−06
  MP6	7.39E−04	0.00E+00	2.78E−02	1.11E−01	1.50E−04	7.17E−04	2.55E−04	6.22E−04	1.15E−04
  2AP	1.17E−07	2.80E−06	4.83E−04	1.53E−03	2.03E−05	1.03E−05	2.50E−07	3.17E−07	0.00E+00
  EMS	0.00E+00	1.67E−07	3.75E−04	8.17E−04	1.10E−04	6.68E−05	0.00E+00	1.33E−06	0.00E+00
  MNNG	0.00E+00	3.67E−07	1.22E−04	5.33E−04	3.22E−05	5.56E−06	3.67E−05	2.44E−07	0.00E+00
  UV	0.00E+00	0.00E+00	5.00E−07	7.49E−06	0.00E+00	0.00E+00	0.00E+00	2.89E−08	0.00E+00
  XL1-Blue	0.00E+00	5.40E−06	0.00E+00	8.66E−07	4.82E−01	0.00E+00	0.00E+00	0.00E+00	9.44E−01
  XL1-Red	0.00E+00	0.00E-00	1.42E−03	2.62E−04	9.84E−01	3.62E−05	3.81E−06	3.60E−05	1.10E−04

• We repeated these antibiotic resistance evolution experiments to compare the performance of these MPs to those of the commonly used chemical mutagens [32] ethyl methanesulfonate (EMS), methylnitronitrosoguanidine (MNNG), and 2-aminopurine (2AP), as well as UV irradiation, XL1-Red, and XL1-Blue (FIGS. 5A, 5B: Table 6). MP6 outperformed all six of the other mutagenesis treatments or strains for all but one of the antibiotics. Cephotaxime resistance was not observed from use of any of the MPs, but was weakly detected from the chemical mutagens (FIG. 6B). We speculate that the known ability of all three of these chemical mutagens to greatly enhance G:C→A:T transitions may contribute to the evolution of cephotaxime resistance. Cumulatively, these results suggest that MP6 can rapidly generate strains with novel properties, outperforming several commonly using chemical mutagens, UV irradiation, and XL-1 Red.
MP6 Allows for Direct RNA Polymerase Evolution During PACE
• Next we compared the performance of MP1 and MP6 during phage-assisted continuous evolution (PACE) [11, 12, 33-35] of T7 RNA polymerase. We previously showed that PACE can evolve T7 RNA polymerase to recognize the distant T3 promoter (P_T3) (FIG. 18), but only using either an intermediate evolutionary stepping-stone (a T7/T3 hybrid promoter) [11, 33, 34], or an initial period of evolutionary drift in the absence of selection pressure [12]. Without either an evolutionary stepping-stone or initial evolutionary drift, PACE cannot evolve T7 RNAP variants that recognize P_T3[11, 12, 33, 34]. We hypothesized that with improved mutagenesis, evolved solutions could be accessed before the phage population washed out without requiring evolutionary stepping-stones or modulated selection stringency.
• We compared the ability of MP1 and MP6 to rapidly evolve P_T3-active T7 RNAP variants in the absence of evolutionary drift, or under conditions in which the selection stringency was slightly reduced. Importantly, previous attempts of T7 RNAP evolution towards P_T3activity using MP1 required a drift period of virtually no selection pressure over 18 h to yield P_T3-active variants, whereas the high or intermediate selection pressures resulted in rapid phage washout 1121.
• In agreement with our previous results, T7 RNAP phage added to lagoons fed by host cells harboring MP1 under high or intermediate selection stringency rapidly washed out in <20 h of PACE when required to recognize P_T3(FIG. 6A). In contrast, both lagoons tested in which host cells harbored MP6 allowed for the propagation of T7 RNAP phage on host cells requiring P_mrecognition under high or intermediate selection stringency, with nearly 10% of both populations after only 10 h of PACE exhibiting activity on P_T3by activity-dependent plaque assays (FIG. 6B). Sequencing confirmed mutations M219K/E222K/E222Q together with N748D in all surviving clones (FIG. 19), in agreement with previous findings of our group and others [11, 12, 33, 34, 36]. The ability of MP6 to support the discovery of P₃-active T7 RNAP variants highlights the ability of this MP to access mutations more efficiently, and thus mediate a more thorough and larger sampling of sequence space. Collectively, these results establish that the enhanced mutagenesis mediated by the new MPs can support accelerated access to evolved proteins that are difficult to access directly using previous methods.
Discussion
• Using a systematic, mechanism-guided approach, we developed a series of vectors that express a variety of genes known to adversely affect DNA replication fidelity. In total we generated and assayed 80 candidate MPs with mutation rates spanning five orders of magnitude (FIGS. 11 and 12; Tables 2 and 3). The resulting MPs support highly potent, broad-spectrum, inducible, vector-based in vivo mutagenesis that rival the performance characteristics of popular in vitro methods such as error-prone PCR, while offering key advantages of in vivo mutagenesis. These advantages include enabling mutation and selection cycles to be coupled, and bypassing transformation efficiency bottlenecks that limit the size of populations that can be generated from DNA diversified in vitro. The MPs developed here offer major advantages over current in vivo mutagenesis methods such as chemical mutagens or base analogs, UV irradiation, or constitutive hypermutator strains. Hypermutator strains, for example, generally suffer from poor transformation efficiency (XL1-Red, ˜1×10⁶cfu/μg plasmid DNA), high instability, and narrow mutagenic spectra. MP6 increases the mutation rate ofE. coliby 322,000-fold, and substantially exceeds both the mutation rate and the mutagenic spectra of XL1-Red. Importantly, MP6 can support approximately ˜2.3 substitutions/kb in a gene of interest using phage vectors in a single generation, with additional increases in mutagenesis efficiency concomitant with longer propagation times.
• To demonstrate the utility of these vectors, we used a whole-genome mutagenesis approach to evolve high-level antibiotic resistance inE. coli. In the absence of any prior selection or mutagenesis step, MP6 rapidly mediates the evolution of antibiotic resistance to many commonly used antibiotics within 18 h. The efficiency and effectiveness of antibiotic resistance mediated by MP6 compares favorably with that of a number of potent chemical mutagens (2AP, EMS, MNNG), UV irradiation, and the hypermutator strain XL1-Red. In addition, we observed that MP6 supports the continuous evolution of 17 RNA polymerase variants capable of initiating transcription at the non-cognate T3 promoter in less than 10 h, without requiring evolutionary stepping-stones or an initial period of evolutionary drift. The MPs provided herein are broadly applicable to the use of in vivo mutagenesis to provide efficient access to rare solutions in sequence space that would otherwise be much more difficult, or impossible, to reach using current in vitro or in vivo methods. The properties of MP6, which include very high mutagenesis efficiency, broad mutational spectrum, small-molecule inducibility, and compatibility with a variety of bacterial strains, together represent a substantial advance in in vivo mutagenesis methodology for the laboratory evolution community.
Materials and Methods
• General methods. All PCR reactions were performed using Pfu Turbo Cx polymerase (Agilent Technologies) or VeraSeq ULtra polymerase (Enzymatics). Water was purified using a MilliQ water purification system (Millipore, Billerica Mass.). All MPs were constructed using USER cloning (New England Biolabs). NativeE. coligenes were amplified by PCR directly from genomic DNA, and non-bacterial genes were synthesized as bacterial codon-optimized gBlocks Gene Fragments (Integrated DNA Technologies). All DNA cloning was carried out using NEB Turbo cells (New England Biolabs).
• General MP strain preparation. Mid log-phase (OD₆₀₀=˜0.5-0.8) cells of the strain of interest grown in 2×YT (United States Biological) were transformed with the desired MP, and recovered for 45 min in Davis rich media¹²to suppress MP induction. All transformations were plated on 2×YT in 1.8% agar (United States Biological) containing 40 μg/mL chloramphenicol (Sigma Aldrich), 10 μg/mL fluconazole (TCI America), 10 μg/mL amphotericin B (TCI America), 25 mM glucose (United States Biological) and grown for 12-18 h in a 37° C. incubator. Colonies transformed with the appropriate MP were picked the following day and grown in Davis rich media containing 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, and 10 μg/mL amphotericin B for 12-18 h. Following overnight growth of the MP-carrying strains, cultures were diluted 1,000-fold into fresh Davis rich media containing 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, and 10 μg/mL amphotericin B. The remainder of each experiment is described in each of the following sections.
• Rifampin resistance assay. Upon reaching mid log-phase, cultures were induced with 25 mM arabinose (Davis rich media+arabinose) or suppressed with 25 mM glucose (Davis rich media only) and allowed to continue growth for an additional ˜18-24 h in a 37° C. shaker. The high arabinose concentration ensures sufficient induction of the plasmid-borne mutators MG1655 ΔrecA::apra despite arabinose catabolism by this strain upon glucose depletion. For XL1-Blue and XL1-Red strains, cultures were started directly from glycerol stocks according to the manufacturer's instructions and incubated for an identical amount of time as the MP-carrying strains. After overnight growth, cultures were serially diluted in 10-fold increments and plated on 2×YT-agar containing 10 μg/mL fluconazole, 10 μg/mL amphotericin B, and 100 mM glucose+/−100 μg/mL rifampin. After 18-24 h, the number of colonies on the glucose+/−rifampin plates was counted for each culture. The mutation efficiency induced by the MP (μ_bp, substitutions/bp/generation) was calculated using the equation: μ_bp=f/[R×ln(N/N₀)], where f is the frequency of rifampin-resistant mutants (as compared to the glucose control), R is the number of sequenced sites yielding rifampin resistance (21 sites across both rpoB clusters in our experiments), N is the final population size, and N₀is the population size at which resistance is first observed (empirically determined to be ˜1.5×10⁷). To calculate μ_G, μ_bpwas multiplied by the genome size, which for MG1655 was 4.64×10⁶bp.
• Episomal lacZ reversion assay. Upon reaching mid log-phase, the cultures were induced with 25 mM arabinose or suppressed with 25 mM glucose, and allowed to continue growth for an additional ˜18-24 h. After overnight growth, the cultures were centrifuged for 2 min at 10,000× rcf and resuspended in an equal volume of 10% glycerol. This procedure was carried out twice to remove trace glucose or other carbon sources from the supernatant prior to plating. Washed cells were serially diluted in 10-fold increments using 10% glycerol and plated on M9 minimal media agar supplemented with 5 mM MgSO₄, 0.01% thiamine, 335 μg/mL Bluo-Gal (Life Technologies) and either 10 mg/mL glucose or 10 mg/mL lactose. The Bluo-Gal was added to ensure that survival on lactose was concomitant with lacZ reversion, and not purely due to extracellular lactose hydrolysis. After extended growth (˜24-36 h), the fraction of lactose-catabolizing colonies was calculated using the number of blue colonies on the lactose plates vs. the total number of colonies on the glucose plates.
• Phage lacZ inactivation assay. Upon reaching mid log-phase, the cultures were induced with 25 mM arabinose or suppressed with 25 mM glucose, allowed to grow for an additional 0-2 h, then, in the case of strain S1030, infected with SP063 phage, and allowed to grow for an additional˜18-24 h. For S1021 and XL1-Red (Agilent Technologies), SP063 DNA was miniprepped from infected S1030 cells and electroporated into these strains as they both lack F′ episomes. For F cells, cultures were either induced for 2 h prior to being made electrocompotent, induced immediately following transformation, induced both prior to and following electroporation, or not induced at all. After overnight growth and phage propagation, the cultures were centrifuged 2 min at 10,000× ref and the supernatant was filtered through a 0.2 μm PVDF filter (Millipore). The supernatant was serially diluted in 10-fold increments using Davis rich media and plaqued on S1030 cells using 1.8% 2×YT-agar for the bottom layer and 0.6% 2×YT-agar supplemented with 400 μg/mL Bluo-Gal (Life Technologies) for the top layer. The fraction of white or light blue plaques (lacZ⁻ phenotype) was counted as a function of all plaques (blue+light blue+white), and used as a measure of mutation frequency for the lacZ cassette.
• Sanger sequencing of rpoB mutations. Rifampin-resistant colonies were picked into 96 well plates and grown overnight in Davis rich media supplemented with 100 μg/mL rifampin. Following overnight growth, 10 μL aliquots were heated at 100° C. for 10 min, followed by PCR using primers AB1678 (5′-AATGTCAAATCCGTGGCGTGAC, SEQ ID NO: 20) and AB1682 (5′-TTCACCCGGATACATCTCGTCTTC, SEQ ID NO: 21) to amplify an rpoB fragment containing bothclusters 1 and II. Each fragment was sequenced twice using primers AB1680 (5′-CGGAAGGCACCGTAAAAGACAT, SEQ ID NO: 22) and AB1683 (5′-CGTGTAGAGCGTGCGGTGAAA, SEQ ID NO: 23).
• High-throughput sequencing of lacZ mutations. SP063 phage that was propagated using S1030 carrying MP1, MP4 or MP6, produced by XL1-Red following SP063 DNA electroporation, or the unmutated stock phage was amplified by PCR using primers AB437 (5′-GGCGCTGGTAAACCATATG, SEQ ID NO: 24) and DB213 (5′-GGAAACCGAGGAAACGCAA, SEQ ID NO: 25) to yield a ˜3,400 bp fragment containing the lacZ gene. SP063 phage that was propagated under similar conditions on S1030 cells was used as the negative control. Three biological replicates were carried out for each of the aforementioned samples. The resulting PCR products were purified by gel electrophoresis using a 1% agarose gel and prepared for HTS using a Nextera kit (Illumina) and a previously described procedure³⁵. Briefly, 4 μL of DNA (2.5 ng/μL), 5 μL TD buffer, and 1 μL TDE1 were mixed together and then heated to 55° C. for 5 min. After purification (Zymo DNA purification kit), the resultant “tagmented” DNA samples were amplified with Illumina-supplied primers using the manufacturer's protocol. The resulting PCR products were then purified using AMPure XP beads and the final concentration of DNA was quantified using PicoGreen (Invitrogen) and qPCR. The samples were sequenced on a MiSeq Sequencer (illumina) in 2×300 paired-end runs using the manufacturer's reagents following the manufacturer's protocols.
• High-throughput sequencing data analysis. A previously described custom MATLAB script³⁵(available upon request) was used to align MiSeq reads with Q score<30 to the wild-type sequence and count the nucleotide positions from which the experimental sample deviates from the wild-type sequence yielding called mutations with >99.9% accuracy, corresponding to >3 s.d, above the mean error rate of the MiSeq high-throughput sequencing reads. To compensate for systemic sample preparation and sequencing errors, the observed fraction of mutations at each nucleotide position of the wild-type lacZ reference gene was subtracted from the fraction of mutations in a given experimental sample to result in the “corrected fraction mutated”. Mutations were defined as nucleotide positions with a corrected fraction mutation that is both greater than the average corrected fraction mutated of the treatment of interest and at least one standard deviation higher than the corrected fraction mutation of the wild-type reference sequence. Duplicates belonging to set of paired-end reads were treated as a single sample, while duplicate reads of the same region with alternative adaptor/index sequences were not removed so as not to introduce bias into the sequencing analysis. This process yielded an average of ˜50,000 reads per position for each of the sequenced samples.
• Evolution of novel antibiotic resistance. MG1655 ΔrecA::apra cells without an MP or carrying MP1, MP4, or MP6 were grown for 18-21 hr in Davis rich media containing 40 μg/mL chloramphenicol, 10 μg/mL fluconazole, 10 μg/mL Amphotericin B, and supplemented with 200 mM arabinose to induce the MPs. Small molecule and UV mutagenesis was carried out as previously described³². For 2AP treatment, log-phase MG1655 ΔrecA::apra cells were diluted to ˜1000 cells, the media was supplemented with 700 μg/mL 2AP (TCI America), and the culture was allowed for grow at 37° C. for an additional 18-21 hr. For EMS treatment, 2 mL of a log-phase MG1655 ΔrecA::apra culture (˜1×10⁸-1×10⁹cells) was centrifuged, washed twice with 1 mL A buffer on ice, then supplemented with 14 μL EMS (TC America). Cells were lightly vortexed, and allowed to shake at 200 rpm at 37° C. for 45 min. After this time, the culture was centrifuged, washed twice with 1 mL A buffer on ice, diluted by 20-fold into Davis rich media without antibiotics, and allowed to grow for 18-21 hr. For MNNG treatment, 2 mL of a log-phase MG1655 ΔrecA::apra culture (˜1×10⁸-1×10⁹cells) was centrifuged, washed twice with 1 mL citrate buffer (pH 5.5) on ice, supplemented with 111 μL of 1 mg/mL MNNG (TCI America) and placed in a 37° C. water bath for 30 min. Following treatment, the cells were centrifuged, washed twice with 1 mL 0.1 M potassium phosphate buffer (pH 7.0), diluted by 4-fold into Davis rich media without antibiotics, and allowed to grow for 18-21 hr. For UV irradiation, 2 mL of a log-phase MG1655 ΔrecA::apra culture (˜1×10⁸-1×10⁹cells) was centrifuged, resuspended in 1 mL 0.1 M MgSO4 and placed on ice for 10 min. Cells were placed in a petri dish and exposed to UV light from a SM-36-2GR UV lamp (American Air & Water) for 1 min, uncovered, at a distance of ˜10 cm. Immediately following UV exposure, cells were diluted by 20-fold into Davis rich media without antibiotics, and allowed to grow for 18-21 hr. For XL1-Blue and XL1-Red strains, cultures were started directly from glycerol stocks according to the manufacturer's instructions and allowed to grow for 18-21 hr in Davis rich media. Following overnight growth, all cultures were serially diluted in Davis rich media and plated on 2×YT-agar containing 10 μg/mL fluconazole, 10 μg/mL amphotericin B, and 100 mM glucose+/−the appropriate antibiotic. After overnight growth (˜18-24 h), the numbers of colonies on the glucose+/−antibiotics plates were counted.
• Continuous evolution of P_T3-active T7 RNAP variants. Two modified versions of MP1 and MP6 (MP1a and MP6a, respectively) were generated to support robust phage propagation during PACE. These MPs carry all of the components of their respective MPs, in addition to the previously described anhydrotetracycline (ATc)-dependent drift promoter driving geneIII¹². S1030 strains carrying either MP in addition to the P_T3accessory plasmid (AP) were inoculated into host-cell cultures (chemostats) and grown at a dilution rate of 1.6 vol/hr as previously described¹². Lagoons flowing from the respective chemostats were maintained at 40 mL, diluted at 0.75 vol/hr, and supplemented with either 25 mM arabinose only (high stringency) or 25 mM arabinose with 30 ng/mL ATc (intermediate stringency) for 8 h prior to infection with packaged T7 RNAP SP. We note that concentrations exceeding 30 ng/mL for extended timeframes during PACE (>24 hr) result in excision of the evolving gene from the selection phage. As continuous flow conditions effectively enrich for SPs capable of rapid replication, selection phage with smaller genomes are rapidly enriched to totally dominate the evolving pool. Each lagoon was infected with 4×10⁹pfu, resulting in an initial titer of 108 pfu/mL of the lagoon. Samples were taken 10 h and 20 h after infection, centrifuged at 10,000 ref for 2 min, then sterile filtered with a 0.2 μm filter and stored overnight at 4° C. Phage aliquots were titered on S1030 cells carrying either the PSP-geneIII AP (total phage) or the P_T3-geneIII AP (P_T3-active phage).
• MP6 optimization. We retained native bacterial ribosome-binding sites (RBSs) upstream of the ORFs for four of the six MP6 genes. The exceptions are dam, which natively lacks a canonical RBS in theE. coligenome, and cda1, which derives from the eukaryoteP. marinusand thus does not use a bacterial Shine-Dalgarno sequence. In an attempt to further enhance mutational potency by modulating the expression of dnaQ926, dam, seqA, emrR, ugi, and cda1, we varied RBS upstream of each of these six genes by individually mutating them to fully complement the 16S rRNA, and resulting in optimal transcript translation. Interestingly, strengthening the RBSs upstream of each of the six genes generally reduced the potency of the MP, with the exception of the seqA and dnaQ926 RBSs (Tables 2 and 3). Strengthening the seqA RBS proved highly lethal under induced conditions, likely as a consequence of impeded genomic replication (Tables 2 and 3). Increasing the strength of the dnaQ926 RBS enhanced the mutagenic potency of the MP by 4-fold under induced conditions, concomitant with a minor increase in background mutagenesis, but was more toxic to bacteria as evidenced by a greater loss of viability under induced conditions (Tables 2 and 3). Additionally, measurement of the mutation rate of the resulting MP became irreproducible, consistent with MP instability under these conditions. These findings together suggest that additional mutagenic potency gains beyond that of MP6 may result in error catastrophe and reduced MP stability.
• LacZ reversion analysis. Strains CSH101 to CSH106 each carry different nonfunctional missense mutants of lacZ at codon 461 (natively encoding glutamic acid) on the F′ episome. If a mutation reverts the nonfunctional codon to a glutamic acid, the strain can synthesize functional LacZ and survives using lactose as the only carbon source. These six strains are designed to report on all 12 possible mutations using this codon reversion. Using these strains, we observed that MP1 had the most narrow episomal mutational spectrum, with a moderate bias towards G:C→A:T (CSH102) and A:T→G:C (CSH106) substitutions (FIG. 17). MP4 showed an improved distribution of mutations, with a still moderate preference for A:T→G:C (CSH106) substitutions (FIG. 18). MP6 showed a near equal distribution of mutations in all six strains, with the exception of A:T→C:G (CSH101) substitutions which were detected at 10- to 100-fold lower levels than the other substitutions (FIG. 16). Taken together, these results suggest that MP6 generally outperforms the other MPs and XL1-Red based on both the frequency of lacZ reversion as well as the breadth of mutation types detected by these strains. Importantly, the mutational potency and spectra using the episomal lacZ reversion assays were in agreement with those from the rifampin resistance assays (FIGS. 4A-E,17). These results establish the ability of the MPs to affect a wide variety of mutations in both genomic and episomal DNA.
Exemplary Mutagenesis Plasmid Sequence
• The Sequence for an exemplary, non-limiting embodiment of a mutagenesis expression construct as provided herein, in this case a mutagenesis plasmid comprising a nucleic acid sequence encoding dnaQ926, dam, seqA, emrR, ugi, and CDA1 is provided below:

• pAB086p10-MP CloDF13 pBAD dnaQ926 dam seqA
  emrR ugi CDA1 cat standard; circular DNA; 6681 BP.

  LOCUS	pJC184 6537 bp DNA circular
  Key	Location/Qualifiers
  terminator	867..911
  	/note=“rrnB1 transcriptional terminator”
  rep_origin	complement(39..777)
  	/dnas_title=“cloDF13”
  	/vntifkey=“33”
  	/label=cloDF13
  terminator	complement (5859..5894)
  	/note=“P14/tonB bidirectional terminator”
  	/note=“termination of cat transcript is slightly weaker
  	than in opposite direction”
  CDS	complement (5908..6567)
  	/note=“cat (CmR)”
  	/note=“from pACYCDuet-1”
  modified_base	5983..5983
  	/note=“mutation”
  	/note=“annotated as a G in pACYCDuet cat marker
  	annotation, here it is an A, this mutation is silent
  	from codon GTC (Val, 25% codon usage)
  	to GTT (Val, 21% usage)”
  promoter	6568..6665
  	/note=“cat promoter”
  	/note=“from pACYCDuet−1”
  CDS	complement (927..1805)
  	/dnas_title=“araC”
  	/vntifkey=“4”
  	/label=araC
  misc_feature	1903..1903
  	/dnas_title=“C to A ***”
  	/vntifkey=“21”
  	/label=C to A ***
  misc_feature	1821..1821
  	/dnas_title=“A to G ***”
  	/vntifkey=“21”
  	/label=A to G ***
  prim_transcript	complement (1969..1969)
  	/note=“pC TSS”
  protein_bind	1992..2008
  	/note=“araO1”
  protein_bind	1971..1987
  	/note=“araO1”
  protein_bind	1834..1850
  	/note=“araO2”
  protein_bind	2013..2034
  	/note=“CAP”
  misc_feature	2093..2093
  	/note=“”
  misc_feature	2079..2079
  	/note=“”
  prim_transcript	2117..2117
  	/note=“pBAD TSS”
  protein_bind	2045..2061
  	/note=“araI1”
  protein_bind	2066..2082
  	/note=“araI2”
  −10_signal	2103..2108
  	/note=“−10”
  −35_signal	2079..2084
  	/note=“−35”
  promoter	2013..2165
  	/dnas_title=“pBAD”
  	/vntifkey=“30”
  	/label=pBAD
  CDS	2166..2897
  	/dnas_title=“dnaQ926”
  	/vntifkey=“4”
  	/label=dnaQ926
  conflict	2199..2201
  	/note=“D12A”
  conflict	2205..2207
  	/note=“E14A”
  RBS	2156..2165
  	/note=“>sd5 RBS”
  RBS	2907..2925
  	/note=“Modified mutS RBS”
  CDS	2926..3762
  	/note=“dam (wt)”
  CDS	3791..4336
  	/note=“seqA (wt)”
  RBS	3771..3790
  	/note=“seqA Native RBS”
  CDS	4365..4895
  	/note=“emrR (wt)”
  RBS	4345..4364
  	/note=“native emrR RBS”
  CDS	4924..5178
  	/note=“PBS2 UGI”
  RBS	4903..4923
  	/note=“Native UGI RBS”
  RBS	5187..5207
  	/note=“dnaE RBS”
  CDS	5208..2834
  	/note=“pmCDA1 (opt)”
  −10_signal	complement (734..739)
  	/note=“−10”
  −35_signal	complement (757..762)
  	/note=“−35”
  −10_signal	(606..611)
  	/note=“−10”
  −35_signal	582..587
  	/note=“−35”
  promoter	complement (729..768)
  	/note=“RNA II Promoter (0.93)”
  promoter	576..615
  	/note=“RNA I Promoter (0.95)”
  misc_RNA	618..722
  	/note=“RNA I”
  misc_RNA	complement (39..725)
  	/note=“RNA II”

• (SEQ ID NO: 26)

  cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt	60
  tacccacaga ttccgtggat aagcagggaa ctaacatgtg aggcaaaaca gcagggccgc	120
  gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt	180
  ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac	240
  ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg	300
  ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc	360
  gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg	420
  ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca	480
  cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg	540
  gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt	600
  ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt	660
  taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg	720
  tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt	780
  tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc	340
  agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg	900
  cctttcgttt tagacttagg gaccctttat gacaacttga cggctacatc attcactttt	960
  tcttcacaac cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc	1020
  gagaaataga gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg	1080
  gaggtgctca aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg	1140
  ctaatcccta actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc	1200
  tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa	1260
  gcctcgcgta cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc	1320
  cgcagtaaca attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct	1380
  tgcccggcgt taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc	1440
  gggcgaaaga accccgtatt ggcaaatatt gacggccagt taagccattc atgccagtag	1500
  gcgcgcggac gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg	1560
  tagtgatgaa tctctcctgg cgggaacagc aaaatatcac ccggtcggca aacaaattct	1620
  cgtccctgat ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc	1680
  attcccagcg gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa	1740
  cccgccacca gatgggcatt aaacgagtat cccggcagca ggggatcatt ttgcgcttca	1800
  gccatacttt tcatactccc accattcaga gaagaaacca attgtccata ttgcatcaga	1860
  cattgccgtc actgcgtctt ttactggctc ttctcgctaa cccaaccggt aaccccgctt	1920
  attaaaagca ttctgtaaca aagcgggacc aaagccatga caaaaacgcg taacaaaagt	1980
  gtctataatc acggcagaaa agtccacatt gattatttgc acggcgtcac actttgctat	2040
  gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc	2100
  gccatagcat ttttatccat aagattagcg gatcctacct gacgcttttt atcgcaactc	2100
  tctactgttt ctccataccc gtttttttgg acgcgtacaa ctcaagtctg acataAAtGA	2160
  ccgctatgag cactgcaatt acacgccaga tcgttctcGC TaccGCAacc accggtatga	2220
  accagattgg tgcgcactat gaaggccaca agatcattga gattggtgcc gttgaagtgg	2280
  tgaaccgtcg cctgacgggc aataacttcc atgtttatct caaacccgat cggctggtgg	2340
  atccggaagc ctttggcgta catggtattg ccgatgaatt tttgctcgat aagcccacgt	2400
  ttgccgaagt agccgatgag ttcatggact atattcgcgg cgcggagttg gtgatccata	2460
  acgcagcgtt cgatatcggc tttatggact acgagttttc gttgcttaag cgcgatattc	2520
  cgaagaccaa tactttctgt aaggtcaccg atagccttgc ggtggcgagg aaaatgtttc	2580
  ccggtaagcg caacagcctc gatgcgttat gtgctcgcta cgaaatagat aacagtaaac	2640
  gaacgctgca cggggcatta ctcgatgccc agatccttgc ggaagtttat ctggcgatga	2700
  ccggtggtca aacgtcgatg gcttttgcga tggaaggaga gacacaacag caacaaggtg	2760
  aagcaacaat tcagcgcatt gtacgtcagg caagtaagtt acgcgttgtt tttgcgacag	2820
  atgaagagat tgcagctcat gaagcccgtc tcgatctggt gcagaagaaa ggcggaagtt	2880
  gcctctggcg aacataattt aatatcagta aaccggacat aacccatgaa gaaaaatcgc	2940
  gcttttttga agtgggcagg gggcaagtat cccctgcttg atgatattaa acggcatttg	3000
  cccaagggcg aatgtctggt tgagcctttt gtaggtgccg ggtcggtgtt tctcaacacc	3060
  gacttttctc gttatatcct tgccgatatc aatagcgacc tgatcagtct ctataacatt	3120
  gtgaagatgc gtactgatga gtacgtacag gccgcacgcg agctgtttgt tcccgaaaca	3180
  aattgcgccg aggtttacta tcagttccgc gaagagttca acaaaagcca ggatccgttc	3240
  cgtcgggcgg tactgttttt atatttgaac cgctacggtt acaacggcct gtgtcgttac	3300
  aatctgcgcg gtgagtttaa cgtgccgttc ggccgctaca aaaaacccta tttcccggaa	3360
  gcagagttgt atcacttcgc tgaaaaagcg cagaatgcct ttttctattg tgagtcttac	3420
  gccgatagca tcgcgcgcgc agatgataca tccgtcgtct attgcgatcc gccttataca	3480
  ccgctgtctg cgaccgccaa ctttacggcg tatcacacaa acagttttac gcttgaacaa	3340
  caagcgcatc tggcggagat cgccgaaggt ctggttgagc gccatattcc agtgctgatc	3600
  tccaatcacg atacgatatt aacgcgtgag tggtatcagc gcgcaaaatt gcatgtcgtc	3660
  aaagttcgac gcagtataag cagcaacggc ggcacacgta aaaaggtgga cgaactgctg	3720
  gctttataca aaccaggagt cgtttcaccc gcaaaaaaat aattcagcta agacactgca	3780
  ctggattaag atgaaaacga ttgaagttga tgatgaactc tacagctata ttgccagcca	3840
  cactaagcat atcggcgaga gcgcatccaa cattttacgg catatgttga aattttccac	3900
  cgcatcacag cctgctgctc cggtgacgaa agaggttcgc gttgcgtcac ctgctatcgt	3960
  cgaagcgaag ccggtcaaaa cgattaaaga caaggttcgc gcaatgcgtg aacttctgct	4020
  ttcggatgaa tacgcagagc aaaagcgagc ggtcaatcgc tttatgctgc tgttgtctac	4080
  actatattct cttgacgccc aggcgtttgc cgaagcaacg gaatcgttgc acggtcgtac	4140
  acgcatttac tttgcggcaa atgaacaaac gctgctgaaa aatggtaatc agaccaagcc	4200
  gaaacatgtg ccaggcacgc cgtattgggt gatcaccaac accaacaccg gccgtaaatg	4260
  cagcatgatc gaacacatca tgcagtcgat gcaattcccg gcggaattga ttgagaaggt	4320
  ttgcaaaact atctaacggc tgaaattaat gaggtcatac ccaaatggat agttcgttta	4380
  cgcccattga acaaatgcta aaatttcgcg ccagccgcca cgaagatttt ccttatcagg	4440
  agatccttct gactcgtctt tgcatgcaca tgcaaagcaa gctgctggag aaccgcaata	4500
  aaatgctgaa ggctcagggA attaacgaga cgttgtttat ggcgttgatt acgctggagt	4560
  ctcaggaaaa ccacagtatt cagccttctg aattaagttg tgctcttgga tcatcccgta	4620
  ccaacacgac gcgtattacc gatgaactgg aaaaacgcgg ttggatcaaa cgtcgtgaaa	4690
  gcgataacga tcgccgctgc ctgcatctgc aattaacgga aaaaggtcac gagtttttgc	4740
  gcgaggtttt accaccgcag cataactgcc tgcatcaact ctggtccgcg ctcagcacaa	4800
  cagaaaaaga tcagctcgag caaatcaccc gcaaattgct ctcccgtctc gaccagatgg	4860
  aacaagacgg tgtggttctc gaagcgatga gctaataata caaaaattag gaggaatttc	4920
  aacatgacaa atttatctga catcattgaa aaagaaacag gaaaacaact agtgattcaa	4980
  gaatcaattc taatgttacc agaagaagta gaggaagtaa ttgggaataa accagaaagt	5040
  gatattttag ttcatactgc ttatgatgaa agtacagatg aaaatgtaat gctattaact	5100
  tcagatgctc cagaatataa accttgggct ttagtaattc aagacagtaa tggagaaaat	5160
  aaaattaaaa tgttataagt cgagattaag taaaccggaa tctgaag	5220
  	5280
  	5340
  	5400
  	5460
  	5520
  	5580
  	5640
  	5700
  	5760
  	5820
  acttaa ttaacggcac tcctcagcca agtcaaaagc ctccgGTcgg	5880
  aggcttttga ctacatgccc atggcgttta cgccccgccc tgccactcat cgcagtactg	5940
  ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg	6000
  aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tagtgaaaac	6060
  ggggacgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca	6120
  gggattggct gagacgaaaa acatattctc aataaaccct ttagagaaat aggccagatt	6180
  ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg	6240
  gtattcactc cagagcgata aaaacgtttc agtttactca tggaaaacgg tgtaacaagg	6300
  gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga actccggatg	6360
  agcattcatc aggcgagcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt	6420
  ctttacggtc tttaaaaagg ccgtaatatc cagctaaacg gtctggttat aggtacattg	6480
  agTaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt	6540
  ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa	6600
  ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac	6660
  gtgccaAgcc aaataggccg t	6690

Exemplary Drift Plasmid Sequences
• The sequences for exemplary, non-limiting embodiments of drift plasmids, named Drift Plasmids 1-6 (DP 1-6), comprising exemplary drift expression constructs, are provided herein. Table 7 shows the coding DNA sequence (CDS) information for exemplary DP plasmids DP1-6.FIGS. 20-25 provide vector maps of drift plasmids DP1-DP6 (SEQ ID NOs: 27-29, 33-35), respectively. It should be understood that mutagenesis plasmids depicted inFIGS. 26-106, corresponding to SEQ ID NOs: 43-123, can be modified to function as drift plasmids by the inclusion of an anhydrotetracycline (ATc)-dependent drift promoter (e.g., SEQ ID NO: 124) in the construct.

• TABLE 7
  Coding DNA sequence (CDS) information for exemplary DP plasmids

  CDS	DP1	DP2	DP3	DP4	DP5	DP6
  araC	2885-3763	2885-3763	2885-3763	2885-3763	2885-3763	2885-3763
  dnaQ926	4124-4855	4124-4855	4124-4855	4124-4855	4124-4855	4124-4855
  umuD	4893-5240	—	—	—	—	—
  umuC	5243-6523	—	—	—	—	—
  recA730	6587-7648	—	—	—	—	—
  dam	—	—	4884-5720	4884-5720	4884-5720	4884-5720
  seqA	—	—	—	5749-6294	5749-6294	5749-6294
  emrR	—	—	—	—	—	6323-6853
  PBS2 UGI	13	—	—	—	6323-6577	6882-7136
  pmCDA1	13	—	—	—	6607-7233	7166-7792

  DP1

  LOCUS  pJC184   6537 bp DNA circular

  FEATURES    Location/Qualifiers

  modified_base 852..866

  /note=“USER junction”

  terminator  867..911

  /note=“rrnB1 transcriptional terminator”

  modified_base 919..933

  /note=“USER junction”

  misc_feature 926..932

  /note=“SanDI”

  /note=“can be used to exchange f1 origin or selection components”

  rep_origin complement(39..777)

  /dnas_title=“cloDF13”

  /vntifkey=“33”

  /label=cloDF13

  terminator complement(7673..7708)

  /note=“P14/tonB bidirectional terminator”

  /note=“termination of cat transcript is slightly weaker

  than in opposite direction”

  modified_base 7709..7721

  /note=“USER junction”

  CDS    complement(7722..8381)

  /note=“cat (CmR)”

  /note=“from pAYCDuet-1”

  modified_base 7797..7797

  /note=“mutation”

  /note=“annotated as a G in pACYCDuet cat marker

  annotation, here it is an A, but this mutation is silent

  from codon GTC (Val, 25% codon usage) to GTT (Val, 21%

  usage), so it should not be of functional relevance”

  misc_feature 7666..7672

  /note=“Nt-BbvCI”

  /note=“nicking endonuclease site that generates overhang”

  modified_base 7649..7665

  /note=“USER junction”

  misc_feature 7651..7658

  /note=“PacI”

  /note=“can be used to exchange antibiotic resistance marker or selection

  components”

  promoter  8382..8479

  /note=“cat promoter”

  /note=“from pACYCDuet-1”

  modified_base 8480..8495

  /note=“USER junction”

  misc_feature 8481..8493

  /note=“SfiI”

  /note=“can be used to exchange plasmid origin or antibiotic resistance marker”

  CDS    complement(2885..3763)

  /dnas_title=“araC”

  /vntifkey=“4”

  /label=araC

  misc_feature 2885..2925

  promoter  complement(3429..3446)

  /dnas_title=“SeqaraC01”

  /vntifkey=“30”

  /label=SeqaraC01

  misc_feature 3861..3861

  /dnas_title=“C to A ***”

  /vntifkey=“21”

  /label=C to A ***

  misc_feature 3779..3779

  /dnas_title=“A to G ***”

  /vntifkey=“21”

  /label=A to G ***

  promoter  3914..4066

  /dnas_title=“pBAD”

  /vntifkey=“30”

  /label=pBAD

  CDS    4124..4855

  /dnas_title=“dnaQ926”

  /vntifkey=“4”

  /label=dnaQ926

  promoter  complement(4353..4371)

  /dnas_title=“dnaQ02”

  /vntifkey=“30”

  /label=dnaQ02

  promoter  complement(4839..4853)

  /dnas_title=“SeqdnaQ03”

  /vntifkey=“30”

  /label=SeqdnaQ03

  CDS    4893..5240

  /dnas_title=“umuD”

  /vntifkey=“4”

  /label=umuD

  CDS    5243..6523

  /dnas_title=“umuC”

  /vntifkey=“4”

  /label=umuC

  CDS    6587..7648

  /dnas_title=“recA730”

  /vntifkey=“4”

  /label=redA730

  misc_feature 7610..7648

  /note=“”

  misc_feature 2936..22936

  /note=“originally an ‘a’ in annotation”

  misc_feature 2975..2975

  /note=“originally a t in annotation”

  misc_feature 3041..3041

  /note=“originaily an a in annotation”

  misc_feature 3245..3245

  /note=“originally a c in annotation”

  misc_feature 3410..3410

  /note=“originally an a in annotation”

  misc_ feature 3569..3569

  /note=“originally a g in annotation”

  misc_ feature 3716..3716

  /note=“originally a g in annotation”

  misc_feature 6925..6925

  /note=“c in originally annotation”

  misc_feature 4051..4051

  /note=“”

  misc_feature 4037..4017

  /note=“”

  primer_bind 39..63

  /note=“AB711”

  primer_bind 164..189

  /note=“AB712”

  primer_bind 3388_3409

  /note=“AB713”

  primer_bind 3266..3287

  /note=“AB714”

  primer_bind 4653..4676

  /note=“AB715”

  primer_bind 4530..4554

  /note=“A9716”

  prime_bind 5945..5968

  /note=“AB717”

  primer_bind 5811_5833

  /note=“AB718”

  primer_bind 7213..7235

  /note=“AB719”

  primer_bind 7079..7101

  /note=“AB720”

  CDS    complement(934..2208)

  /dnas_title=“III”

  /vntifkey=“4”

  /label=III

  misc_feature complement(1021..1066)

  /note=“modified to remove internal promoter”

  primer_bind 1060..1085

  /note=“AB691”

  primer_bind 1234..1254

  /note=“AB424”

  primer_bind 1094..1114

  /note=“AB423”

  misc_feature 2423..2435

  /note=“USER linker”

  RBS    complement(2209..2222)

  /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.

  1992)”

  modified_base complement (2223..2237)

  /note=“USER junction”

  misc_feature complement(2229..2236)

  /note=“SbfI”

  misc_feature complement(2224..2229)

  /note=“BsgI”

  misc_feature complement(2317..2324)

  /note=“”

  misc_feature complement(2308..2312)

  /note=“”

  misc_ feature complement(2297..2297)

  /note=“”

  primer_bind complement(2305..2345)

  /note=“”

  primer_bind 2266..2324

  /note=“”

  protein_bind complement(2278..2296)

  /note=“tetR binding site”

  terminator complement(2443..2867)

  /note=“rrnB1 transcriptional terminator”

  primer_bind 2489..2511

  /note=“AB428”

  primer_bind 2240..2267

  /note=“AB721”

  misc_feature 2868..2884

  /note=“USER linker”

  modified_base 789..806

  /note=“User Junction”

  primer_bind 789..818

  /note=“”

  primer_bind complement(2847..2884)

  /note“”

  primer_bind 2868..2912

  /note“”

  primer_bind complement(777..806)

  /note“”

  misc_difference 912..918

  /note=“cloning scar”

  source   1..8495

• (SEQ ID NO: 27)

  1	cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt
  61	tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc
  121	gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt
  181	ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac
  241	ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg
  301	ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc
  361	gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg
  421	ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca
  481	cttgaatcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg
  541	gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt
  601	ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt
  661	taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg
  721	tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
  781	tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
  841	agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg
  901	cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt
  961	agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc
  1021	gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa
  1081	gacaaaaggg cgacattcaa ccgattgagg gagggaaggt aaatattgac ggaaattatt
  1141	cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa
  1201	atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat
  1261	agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc
  1321	gttttcatcg gcattttcgg tcataacccc cttattagcg tttgccatct tttcataatc
  1381	aaaatcaccg gaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga
  1441	accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc
  1501	gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa
  1561	tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc
  1621	gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt
  1681	gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt
  1741	attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc
  1801	ggggttttgc tcagtaccag gcggataagt gccgtcgaga aagttgatat aagtatagcc
  1861	cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc
  1921	ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg
  1981	aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtaacatt
  2041	ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt
  2101	agtaaatgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cdgcggagtg
  2161	agaatagaaa agaacaacta aaggaattgc gaataataat tttttcattt tttttttcct
  2221	ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  1741	attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc
  1801	ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc
  1861	cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc
  1921	ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg
  1981	aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt
  2041	ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt
  2101	agtaaatgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cagcggagtg
  2161	agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct
  2221	ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  2281	ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga
  2341	tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca
  2401	ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa
  2461	aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt catggcgggc
  2521	gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat
  2581	ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
  2641	tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
  2701	cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
  2761	accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat
  2821	ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga
  2881	ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
  2941	cgctcgggct ggccccggtg catttttcaa atacccgcga gaaatagagt tgatcgtcaa
  3001	aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
  3061	cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
  3121	aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
  3181	aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
  3241	tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
  3301	gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
  3361	caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
  3421	caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
  3481	actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
  3541	ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
  3601	cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
  3661	aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
  3721	acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac
  3781	cattcagaga agaaaccaat tgtccatact gcatcagaca ttgccgtcac tgcgtctttt
  3841	actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
  3901	gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
  3961	tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
  4021	gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
  4081	ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac
  4141	acgccagatc gttctcgcta ccgcaaccac cggtatgaac cagattggtg cgcactatga
  4201	aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa
  4261	taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca
  4321	tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt
  4381	catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt
  4441	tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa
  4501	ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga
  4561	tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact
  4621	cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc
  4681	ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt
  4741	acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga
  4801	agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataaaccgg
  4861	tatgcctcac acaggaaaca gaattcatta ccatgggctt tccttcaccg gcagcagatt
  4921	acgttgaaca gcgcatcgat ctgaatcaac tgttgatcca gcatcccagc gcgacttact
  4981	tcgtcaaagc aagtggtgat tctatgattg atggtggaat tagtgacggt gatttactga
  5041	ttgtcgatag cgctattacc gccagccatg gtgatattgt catcgctgct gttgacggcg
  5101	agtttacggt gaaaaaattg caactacgcc cgacggtaca gcttattccc atgaacagcg
  5161	cgtactcgcc cattaccatc agtagtgaag atacgctgga tgtctttggt gtggtgatcc
  5221	acgtcgttaa ggcgatgcgc tgatgtttgc cctctgtgat gtaaacgcgt tttatgccag
  5281	ctgtgagacg gtgtttcgcc ctgatttatg gggtaaaccg gtggttgtgc tatcgaataa
  3341	tgacggttgc gttatcgccc gaaacgctga ggcaaaggcg cttggcgtta aaatgggcga
  5401	tccctggttc aaacaaaaag atctgtttcg tcgctgtggc gtggtttgct ttagcagcaa
  5461	ttatgagctt tacgcagaca tgagcaatcg ggtgatgtcg acgctggaag agctatcgcc
  5521	ccgcgtcgag atttacagta ccggtatgcc tattgatgag gcattctgcg atctgacagg
  5581	tatgcgtaat tgtcgcgatc tgactgattt tggcagagaa attcgcgcaa cggtgctaca
  5641	acgtacccat cttactgttg gtgtggggat cgcccagacc aaaacgctgg ctaagcttgc
  5701	caatcatgcg gcaaaaaaat ggcagcggca gacgggtggg gtggtggatt tatcaaatct
  5761	ggaacgccag cgtaaattaa tgtctgccct ccccgtggat gacgtctggg ggattggacg
  5821	gcggatcagc aaaaaactgg acgcgatggg gatcaaaacc gttctcgatt tggcggatac
  5881	agatatccgg tttatccgta aacattttaa tgtcgtgctc gaaagaacgg tgcgtgaact
  5941	gcgcggcgaa ccctgtttgc aactggaaga gtttgcaccg acgaagcagg aaattatctg
  6001	ttcccgctcg tttggtgaac gcatcacgga ttatccgtcg atgcggcagg ccatttgtag
  6061	ttacgctgcc cgggcggcgg aaaaacttcg cagcgagcat caatattgtc gattaatttc
  6121	cacgtttatt aagacgtcac catttgcgct caatgaacct tattacggca atagcgcgtc
  6181	ggtaaaactg ctgacgccca ctcaggacag cagggatatc attaacgctg ctacgcgatc
  6241	tctggatgcc atctggcaag cgggccatcg ttaccaaaaa gcgggcgtga tgctggggga
  6301	tttcttcagt cagggagtcg cgcagctcaa tttattcgat gacaacgcac cgcgccccgg
  6361	gagtgagcaa ttgatgacgg taatggatac actgaatgct aaagagggca gaggaacact
  6421	ctattttgcc gggcagggga tccagcaaca atggcagatg aagcgagcca tgctttcacc
  6481	acgttataca acgcgaagtt ctgatttact gagggtcaaa taaatatagc ggcaggaaaa
  6541	aagcgatccc gcatatccgg tattacccgg catgacagga gtaaaaatgg ctatcgacga
  6601	aaacaaacag aaagcgttgg cggcagcact gggccagatt gagaaacaat ttggtaaagg
  6661	ctccatcatg cgcctgggtg aagaccgttc catggatgtg aaaaccatct ctaccggttc
  6721	gctttcactg gatatcgcgc ttggggcagg tggtctgccg atgggccgta tcgtcgaaat
  6781	ctacggaccg gaatcttccg gtaaaaccac gctgacgctg caggtgatcg ccgcagcgca
  6841	gcgtgaaggt aaaacctgtg cgtttatcga tgctgaacac gcgctggacc caatctacgc
  6901	acgtaaactg ggcgtcgata tcgataacct gctgtgctcc cagccggaca ccggcgagca
  6961	ggcactggaa atctgtgacg ccctggcgcg ttctggcgca gtagacgtta tcgtcgttga
  7021	ctccgtggcg gcactgacgc cgaaagcgga aatcgaaggc gaaatcggcg actctcacat
  7081	gggccttgcg gcacgtatga tgagccaggc gatgcgtaag ctggcgggta acctgaagca
  7141	gtccaacacg ctgctgatct tcatcaacca gatccgtatg aaaattggtg tgatgttcgg
  7201	taacccggaa accactaccg gtggtaacgc gctgaaattc tacgcctctg ttcgtctcga
  7261	catccgtcgt atcggcgcgg tgaaagaggg cgaaaacgtg gtgggtagcg aaacccgcgt
  7321	gaaagtggtg aagaacaaaa tcgctgcgcc gtttaaacag gctgaattcc agatcctcta
  7381	cggcgaaggt atcaacttct acggcgaact ggttgacctg ggcgtaaaag agaagctgat
  7441	cgagaaagca ggcgcgtggt acagctacaa aggtgagaag atcggtcagg gtaaagcgaa
  7501	tgcgactgcc tggctgaaag ataacccgga aaccgcgaaa gagatcgaga agaaagtacg
  7561	tgagttgctg ctgagcaacc cgaactcaac gccggatttc tctgtagatg atagcgaagg
  7621	cgtagcagaa actaacgaag atttttaaac ttaattaacg gcactcctca gccaagtcaa
  7681	aagcctccga ccggaggctt ttgactacat gcccatggcg tttacgcccc gccctgccac
  7741	tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
  7801	gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
  7861	cccatagtga aaacgggggc gaagaagtcg tccatattgg ccacgtttaa atcaaaaccg
  7921	gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa cccLttaggg
  7981	aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
  8041	cggaaatcgt cgtagtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
  8101	acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
  8161	cggaactccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
  8221	ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
  8281	ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
  8341	gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
  8401	gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
  8461	ttggaacctc ttacgtgcca Agccaaatag gccgt

  (SEQ ID NO: 28)

  1	cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt
  61	tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc
  121	gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt
  181	ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac
  241	ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg
  301	ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc
  361	gcttcctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg
  421	ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca
  481	cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg
  541	gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt
  601	ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt
  661	taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg
  721	tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
  781	tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
  841	agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg
  901	cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt
  961	agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc
  1021	gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa
  1081	gacaaaaggg cgacattcaa ccgattgagg gagggaaggt aaatattgac ggaaattatt
  1141	cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa
  1201	atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat
  1261	agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc
  1321	gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc
  1381	aaaatcaccg gaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga
  1441	accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc
  1501	gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa
  1561	tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc
  1621	gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt
  1681	gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt
  1741	attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc
  1801	ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc
  1861	cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc
  1921	ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg
  1981	aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt
  2041	ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgct
  2101	agtaaatgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cagcggagtg
  2161	agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct
  2221	ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  2281	ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga
  2341	tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca
  2401	ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa
  2461	aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc
  2521	gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat
  2581	ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
  2641	tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
  2701	cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
  2761	accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat
  2821	ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga
  2881	ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
  2941	cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
  3001	aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
  3061	cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
  3121	aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
  3181	aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
  3241	tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
  3301	gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
  3361	caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
  3421	caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
  3481	actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
  3541	ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
  3601	cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
  3661	aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
  3721	acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac
  3781	cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
  3841	actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
  3901	gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
  3961	tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
  4021	gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
  4081	ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac
  4141	acgccagatc gttctcgcta ccgcaaccac cggtatgaac cagattggtg cgcactatga
  4201	aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa
  4261	taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca
  4321	tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt
  4381	catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt
  4441	tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa
  4501	ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga
  4561	tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact
  4621	cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc
  4681	ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt
  4741	acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga
  4801	agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataaactta
  4861	attaacggca ctcctcagcc aagtcaaaag cctccgaccg gaggcttttg actacatgcc
  4921	catggcgttt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt
  4981	ctgccgacat agaagccatc acaaacagca tgatgaacct aaatcgccag cggcatcagc
  5041	accttatcgc cttgcgtata atatttggcc atagtgaaaa cgagggcgaa gaagttgtcc
  5101	atattggcca cgtttaaatc aaaactgatg aaactcaccc agggattggc tgagacgaaa
  5161	aacatattct caataaaccc tttagaaaaa taggccaggt tttcaccgta acacgccaca
  5221	tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat
  5281	gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc
  5341	accagctcac cgtctttcat tgccatacga aactccagat aagcaticat caggcaagca
  5401	agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag
  5461	gccgtaatat ccagctgaac ggtctgatta taggtacatt gagcaactga ctgaaatgcc
  5521	tcaaaatatt ctttacgatg ccattaagat atatcaacgg tagtatgatc agtgattttt
  5581	ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt
  5641	agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccaAgc caaataggcc
  5701	gt

  (SEQ ID NO: 29)

  1	cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt
  61	tacccacaga ttccgtggat aagcaaagga ctaacatgtg aagcaaaaca gcaggaccgc
  121	gccggtgacg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt
  181	ttccggtgca tctgtaggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac
  241	ccgacaggac ttaaagatcc ccaccctttc cggcggatcg ctccctcttg cgctctcctg
  301	ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc
  361	gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg
  421	ggctgtaagc aagaactccc cattcaaccc gactgctgcg ccttatccgg taactcttca
  481	cttaaatcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg
  541	gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt
  601	ccggctacac tggaaggaca gatttgcttg ctgtgctctg caaaagccag ttaccacggt
  661	taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt gattttttcg
  721	tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
  781	tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
  841	agcccaggag gaagaggaca tccgatcaaa taaaacgaaa ggctcagtcg aaagactggg
  901	cctttcgtgg tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt
  961	agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc
  1021	gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa
  1081	gacaaaaggg cgacattcaa ccgattgagg gaaagaaggt aaatattgac ggaaattatt
  1141	cattaaaggt gaattatcac cgtcaccgac ttgagccatt taggaattag agccagcaaa
  1201	atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat
  1261	agcagcaccg taatcagtaa cgacagaatc aactttgcct ttagcgtcaa actgtagcgc
  1321	gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc
  1381	aaaatcaccg gaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga
  1441	accaccaccc tcagagccac caccctcaga gccaccacca gaaccaccac cagagccgcc
  1501	gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa
  1561	tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc
  1621	gtcatacatg gcttttgata atacaggagt gtactggtaa taagttttaa cgaggtcagt
  1681	gccttgagta acaatgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt
  1741	attctgaaac atgaaagtat taagaggctg agactcctca agagaaggat taggattagc
  1801	ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc
  1861	cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc
  1921	ctcagaaccg ccaccctcag agccaccacc ctcattttca gggatagcaa gcccaatagg
  1981	aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt
  2041	ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt
  2101	agtaaatgaa ttttctgtat gaggttttgc taaacaactt tcaacagttt cagcgaagtg
  2161	agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct
  2221	ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  2281	ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga
  2341	tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca
  2401	ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa
  2461	aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tataacaaac
  2521	gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cttggcggat
  2581	ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
  2641	tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
  2701	cccacactac catcgacgct acggcgtttc acttctgagt tcggcatggg gtcagatgga
  2761	accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat
  2821	ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga
  2881	ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacgaaact
  2941	cgctcgggct ggccccaatg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
  3001	aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
  3061	cctggctgat acgttcggtc tcgcgccagc ttaagacgct aatccctaac tgctgacgga
  3121	aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgcta acgatatcaa
  3181	aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
  3241	tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
  3301	gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
  3361	caaacaggtc gctgaaatgc gactggtgcg cttcatccgg gcgaaagaac cccgtattgg
  3421	caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
  3481	actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
  3541	ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
  3601	cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
  3661	aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
  3721	acgagtatcc cggcagcaaa ggatcatttt gcacttcagc catacttttc atactcccac
  3781	cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcatctttt
  3841	actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
  3901	gcaagaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
  3961	tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
  4021	gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
  4081	ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac
  4141	acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattgatg cgcactatga
  4201	aggccacaag atcattgaga ttgatgccgt tgaagtggtg aaccgtcgcc tgacgggcaa
  4261	taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca
  4321	tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt
  4381	catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt
  4441	tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa
  4501	ggtcaccgat agccttgcgg tggcaaggaa aatgtttccc ggtaagcgca acagcctcga
  4561	tgcgttatgt actcgctacg aaataaataa cagtaaacga acgctgcacg gggcattact
  4621	cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc
  4681	ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt
  4741	acgtcaggca agtaagttac gcgttgtttt tgcgacagat aaagagattg cagctcatga
  4801	agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa
  4861	tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaag tgggcagggg
  4921	gcaagtatcc cctgcttgat gatattaaac ggcatttgcc caagggcgaa tgtctggttg
  4981	agccttttgt aggtgccggg tcggtgtttc tcaacaccga cttttctcgt tatatccttg
  5041	ccgatatcaa tagcgacctg atcagtctct ataacattgt gaagatgcgt actgatgagt
  5101	acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc
  5161	agttccgcga agagttcaac aaaagccagg atccgttccg tcgggcggta ctgtttttat
  5221	atttgaaccg ctacggttac aacggcctgt gtcgttacaa tctgcgcggt gagtttaacg
  5281	tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg
  5341	aaaaagcgca gaatgccttt ttctattgtg agtcttacgc cgatagcalg gcgcgcgcag
  5401	atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact
  5461	ttacggcgta tcacacaaac agttttacgc ttgaacaaca agcgcatctg gcggagatcg
  5521	ccgaaggtct ggttgagcgc catattccag tgctgatctc caatcacgat acgatgttaa
  5581	cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca
  5641	gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg
  5701	tttcacccgc gaaaaaataa acttaattaa cggcactcct cagccaagtc aaaagcctcc
  5761	gaccggaggc ttttgactac atgcccatgg cgtttacgcc ccgccctgcc actcatcgca
  5821	gtaccgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg
  5881	aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatagt
  5941	gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact
  6001	cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc
  6061	caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc
  6121	gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta
  6181	acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaactc
  6241	cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt
  6301	atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt
  6361	acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc
  6421	aacggtggta tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct
  6481	cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc
  6541	tcttacgtgc caAgccaaat aggccgt

  (SEQ ID NO: 33)

  1	cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt
  61	tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc
  121	gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt
  181	ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac
  241	ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg
  301	ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc
  361	gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg
  421	ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca
  481	cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg
  541	gagtccgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt
  601	ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt
  661	taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg
  721	tttacagggc aaaagattac gcgcagaaaa aaaggacctc aagaagatcc cttgatcttt
  781	tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
  841	agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg
  901	cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt
  961	agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc
  1021	gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggtttac cagcgccaaa
  1081	gacaaaaggg cgacattcaa ccgattgagg gagggaaggt aaatattgac ggaaattatt
  1141	cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa
  1201	atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcgat
  1261	agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc
  1321	gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc
  1381	aaaatcaccg aaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga
  1441	accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc
  1501	gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa
  1561	tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc
  1621	gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt
  1681	gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt
  1741	attctgaaac atgaaagtat taagaggctg agactcctca aaagaaggat taggattagc
  1801	gggattttgc tcagtaccag acggataagt gccgtcgaga gggttgatat aagtatagcc
  1861	Aggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc
  1921	ctcagaaccg ccaccctcag agccaccacc ctcattttca aagatagcaa gcccaatagg
  1981	aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt
  2041	ccacagacag ccctcatagt tagcgtaacg atctaaagtt ttgtcgtctt tccagacgtt
  2101	agtaaatgaa ttttctgtat gggattttgc taaacaactt tcaacagttt cagcggagtg
  2161	agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct
  2221	ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  2281	ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga
  2341	tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca
  2401	ctaacaagtg gcgaatttca tcacggagcc aatatcctac gcgagtttat agaaacgcaa
  2461	aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc
  2521	gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat
  2581	ttatcctact caggagagca ttcaccgaca aacaacagat aaaatgaaaa gcccagtctt
  2641	tcgactgagc cccccgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
  2701	cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
  2761	accaccgcgc tactgccgcc aggcaaattc tattttatca gaccgcttct gcgttctgat
  2821	ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga
  2881	ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
  2941	cgctcgagct ggccccggtc cattttttaa atacccgcga gaaatagagt tgatcgtcaa
  3001	aaccaacatt gcgaccgacg gtggcgatag gcatccgggt gatactcaaa agcagcttcg
  3061	cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
  3121	aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacactg gcgatatcaa
  3181	aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
  3241	tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
  3301	gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcatta atgatttgcc
  3301	caaacaggtc gctgaaatgc ggctgatacg cttcatccgg gcaaaagaac cccgtattgg
  3421	caaatattga cggccagtta agccattcat gccagtaggc acgcggacga aagtaaaccc
  3481	actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
  3541	ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
  3601	cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcaataa
  3661	aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
  3721	acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac
  3781	cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
  3841	actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
  3901	gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
  3961	tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
  4021	gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
  4081	ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac
  4141	acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattggtg cgcactatga
  4201	aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa
  4261	taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca
  4321	tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt
  4381	catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt
  4441	tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa
  4501	ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga
  4561	tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact
  4621	cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc
  4681	ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt
  4741	acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga
  4801	agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa
  4861	tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaag tgggcagggg
  4921	gcaagtatcc cctgcttgat gatattaaac ggcatttgcc caagggcgaa tgtctggttg
  4981	agccttttgt aggtgccggg tcggtgtttc tcaacaccga ctcttctcgt tatatccttg
  5041	ccgatatcaa tagcgacctg atcagtctct ataacattgt gaagatgcgt actgatgagt
  5101	acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc
  5161	agttccgcga agagttcaac aaaagccagg atccgttccg tcgggcggta ctgtttttat
  5221	atttgaaccg ctacggttac aacggcctgt gtcgttacaa tctgcgcggt gagtttaacg
  5281	tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg
  5341	aaaaagcgca gaacgccttt ttctattgtg agtcttacgc cgatagcatg gcgcgcgcag
  5401	atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact
  5461	ttacggcgta tcacacaaac agttttacgc ttgaacaaca agcgcatctg gcggagatcg
  5521	ccgaaggtct ggttgagcgc catattccag tgctgatctc caatcacgat acgatgttaa
  5581	cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca
  5641	gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg
  5701	tttcacccgc gaaaaaataa ttcagctaag acactgcact ggattaagat gaaaacgatt
  5761	gaagttgatg atgaactcta cagctatatt gccagccaca ctaagcatat cggcgagagc
  5821	gcatccgaca ttttacggcg tatgttgaaa ttttccgccg catcacagcc tgctgctccg
  5881	gtgacgaaag aggttcgcgt tgcgtcacct gctatcgtcg aagcgaagcc ggtcaaaacg
  5941	attaaagaca aggttcgcgc aatgcgtgaa cttctgcttt cggatgaata cgcagagcaa
  6001	aagcgagcgg tcaatcgctt tatgctgctg ttgtctacac tatattctct tgacgcccag
  6061	gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac gcgtttactt tgcggcagat
  6121	gaacaaacgc tgctgaaaaa tggtaatcag accaagccga aacatgtgcc aggcacgccg
  6181	tattgggtga tcaccaacac caacaccggc cgtaaatgca gcatgatcga acacatcatg
  6241	cagtcgatgc aattcccggc ggaattgatt gagaaggttt gcggaactat ctaaacttaa
  6301	ttaacggcac tcctcagcca agtcaaaagc ctccgaccgg aggcttttga ctacatgccc
  6361	atggcgttta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc
  6421	tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca
  6481	ccttgtcgcc ttgcgtataa tatttgccca tagtgaaaac gggggcgaag aagttgtcca
  6541	tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa
  6601	acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat
  6661	cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg
  6721	aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca
  6781	ccagctcacc gtctttcatt gccatacgga actccggatg agcattcatc aggcgggcaa
  6841	gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg
  6901	ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct
  6961	caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt
  7021	tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
  7081	gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccaAgcc aaataggccg
  7141	t

  (SEQ ID NO: 34)

  1	cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc actgcggaca catacaaagt
  61	tacccacaga ttccgtggat aagcagagga ctaacatgtg aggcaaaaca gcagggccgc
  121	gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt
  181	ttccggtgca tctgtgagag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac
  241	ccgacaggac ttaaagatcc ccgccgtttc cggcggatcg ctccctcttg cgctctcctg
  301	ttccgaggct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc
  361	gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttca ctccaagctg
  421	ggctgtaagc aagaactccc cattcaggcc gactgctgcg ccttatccgg taactgttca
  481	cttgagtgca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg
  541	gaattcgcag aggatttgtt tagctaaaca cgcggttact cttgaagtct gcgccaaagt
  601	ccggctacac tggaaggaca gatttggttg ctgtagtctg cgaaagccag ttgccacggt
  661	taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg
  721	tttacagggc aaaagattac gcgcagaaaa aaaagatctc aagaagatcc tttgatcttt
  781	tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
  841	agcccaggag gaagaggaca tccgatcaaa taaaacgaaa ggctcagtcg aaagactggg
  901	cctttcattt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt
  961	agcaaacgta gaaaatacat acataaaaat ggcaacatat aaaagaaacg caaagacacc
  1021	gcggaacagg ttgatcttat cgcagtcgat actggactcg taaggtttac cagcgccaaa
  1031	gacaaaaggg cgacattcaa ccgattgagg gaaagaaggt aaatattgac ggaaattatt
  1141	cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa
  1201	atcaccagta acaccattac cattaacaag gccggaaacg tcaccaatga aaccatcgat
  1261	agcagcaccg taatcagtag cgacaggatc aagtttgcct ttagcgtcag actgtagcgc
  1321	gttttcgtcg gcattttcgg tcgtagcccc cttattagcg tttgccatct tttcataatc
  1381	aaaatcaccg aaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga
  1441	accgccaccc tcagggccac caccctcaga gccgccacca gaaccaccac cagagccgcc
  1501	gccagcgttg gcgggaggtt ggggcgggtc agacgattgg ccttgatatt cacaaacgaa
  1561	tggatcctca ttaaagccag gatggaaagc gcagtttttg aatttaccgt tccagtaagc
  1621	gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cggggtcagt
  1681	gccttgagta acggtgcccg tgtggacagt taatgccccc tgcctatttc ggaacctatt
  1741	attctgaaac atgaaagtat taagaaactg agactcctca aaagaaggat taggattagc
  1801	ggggttttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc
  1861	cggaatgggt gtatcaccgt actcaggagg tttagtgccg ccaccctcag aaccgccacc
  1921	ctcaggaccg ccaccctcag agccaccacc ctcattttca gggatagcag gtccaatagg
  1981	aacccatgta ccgtaacact gagtttcgtc accagtacaa actattaacg ctgtagcatt
  2041	ccacagacag ccctcatagt tagcgtaacg atctaaggtt ttgtcgtctt tccagacgtt
  2101	agtaggtgaa ttttctgtat ggggttttgc taaacaactt tcaacagttt cagcggagtg
  2161	agaatagaaa ggaattaact aaggaattgc gaataataat tttttcattt tttttttcct
  2221	ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  2281	ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atcttttcga
  2341	tttataaaga tctgattgag gaatcaacag caacatgcca ggatgagtta gcgaattaca
  2401	ctaacaagtg gcgaatttca tcacggagcc aatatcctca gcgagtttat agaaacgcaa
  2461	aaaggccgtc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcaggc
  2521	gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat
  2581	ttatcctact caggagagca ttcaccgaca aacaacagat aaaacgaaaa gcccagtctt
  2641	tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
  2701	cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
  2761	accaccgcgc tactgccgcc aggcaaattc tgttttattc gaccgcttct gcgttctgat
  2821	ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga
  2881	ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
  2941	cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
  3001	aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
  3061	cctggctgat acgttggtcc tcgcgccagc ttaagacgct actccctaac tgctggcgga
  3121	aaagatgtga cagacgcgac ggcgacaggc aaacatgctg tgcggcgctg gcgatatcaa
  3181	aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
  3241	tcggtggatg aagcgactcg ttaatcactt ccatgcgccg cagtaacaat tgctcaagca
  3301	gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
  3361	caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
  3421	caaatattga cggccagtta agccattcat gccagtaggc acgcggacga aagtaaaccc
  3481	actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
  3541	ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
  3501	cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
  3661	aaaaattgag ataaccgttg gcctcaatcg gcgttaaacc caccaccaga tgggcattaa
  3721	gcgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac
  3781	cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
  3841	actggctctt ctcgctaacc caaccaataa ccccgcttat taaaagcatt ctgtaacaaa
  3901	gcgagaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
  3951	tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
  4021	gattggcgga tcctacctga cgctttttat cgcagctctc tactgtttct ccatacccgt
  4081	ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagga ctgcaattac
  4141	acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattggtg cgcactatga
  4201	aggccacaag atcattgaga ttggtaccgt tgaagtggtg aaccgtcgcc tgacgaacaa
  4261	taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca
  4321	tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt
  4381	catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt
  4441	tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa
  4501	ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga
  4561	tgcgttatgt gctcgctacg aaatagataa cagtaaacga acgctgcacg gggcattact
  4621	cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc
  4631	ttttgcgatg gaaggagaaa cacaacagca acaaggtgaa gcaacaattc agcgcattgt
  4741	acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga
  4801	agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa
  4861	tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaaa tgggcagggg
  4921	gcaagtatcc cctgcttgat ggtattaaac ggcatttgcc caagggcgaa tgtctggttg
  4981	agccttttgt aggtgccggg tcggtgtttc tcaacaccga cttttctcgt tatatccttg
  5041	ccaatatcaa tagcgaccta atcagtctct ataacattgt gaagatgcat actgatgagt
  5101	acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc
  5161	agttccgcga agagttcagc aaaagccagg atccgttccg tcgggcggta ctgtttttat
  5221	atttgaaccg ctacggttac aacggcctgt gtcattacaa tctgcgcgat gagtttagcg
  5281	tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg
  5341	aaaaagcgca gaatgccttt ttctattgtg agtcttacgc cgatagcatg gcgcgcgcag
  5401	atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact
  5461	ttacggcgta tcacacaaac agttttacgc ttgaacaaca agcgcatctg gcggagatcg
  5521	ccgaaggtct ggttgagcgc catattccag tgctggtctc caatcacgat gcgatgttaa
  5581	cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca
  5641	gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg
  5701	tttcgcccgc aaaaaaataa ttcagctaag acactgcact aaattaagat gaaaacaatt
  5761	gaagttgatg atgaacycta cagctatatt gccagccaca ctaagcatat cggcgagagc
  5821	gcatccgaca ttttacggcg tatgttgaaa ttttccgccg catcacagcc tgctgctccg
  5881	gtgacgaaag aggttcgcgt tgcgtcacct gctatcgtcg aagcgaagcc ggtcaaaacg
  5941	attaaagaca aggttcgcgc aatgcgtgaa cttctgcttt cggatgaata cgcagagcaa
  5001	aagcgagcgg tcaatcgctt tatgctgctg ttgtctacac tatattctct tgacgcccag
  6001	gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac acatttactt tgcggcagat
  6061	gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac gcgtttactt tgcggcagat
  6121	gaacaaacgc tgctgaaaaa tggtaatcag accaagccga aacatgtgcc aggcacgccg
  6181	tattgggtga tcaccaacac caacaccggc cgtaaatgca gcatgatcga acacatcatg
  6241	cagtcgatgc aattcccggc ggaattgatt gagaaggttt gcggaactat ctaataatac
  6301	aaaaattagg aggaatttca acatgacaaa tttatctgac atcattgaaa aagaaacagg
  6361	aaaacaacta gtgattcaag aatcaattct aatgttacca gaagaagtag aggaagtaat
  6421	tgggaataaa ccagaaagtg atattttagt tcatactgct tatgatgaaa gtacagatga
  6481	aaatgtaatg ctattaactt cagatgctcc agaatataaa ccttgggctt tagtaattca
  6541	sgacagtaat ggagaaaata aaattaaaat gttataagtc gagattaagt aaaccggaat
  6601	ctgaagatga ccgacgcgga atacgttcgt atccacgaaa aactggacat ctacaccttc
  6661	aaaaaacagt tcttcaacaa caaaaaatct gtttctcacc gttgctacgt tctgttcgaa
  6721	ctgaaacgtc gtggtgaacg tcgtgcgtgc ttctggggtt acgcggttaa caaaccgcag
  6781	tctggtaccg aacgtggtat ccacgcggaa atcttctcta tccgtaaagt cgaagaatac
  6841	ctgcgtgaca acccgggtca gttcaccatc aactggtact cttcttggtc tccgtgcgcg
  6901	gactgcgcgg aaaaaatcct ggaatggtac aaccaggaac tgcgtggtaa cggtcacacc
  6961	ctgaaaatct gggcgtgcaa actgtactac gaaaaaaacg cgcgtaacca gatcggtctg
  7021	tggaacctgc gtgacaacgg tgttggtctg aacgttatgg tttctgaaca ctaccagtgc
  7081	tgccgtaaaa tcttcatcca gtcttctcac aaccagctga acgaaaaccg ttggctggaa
  7141	aaaaccctga aacgtgcgga aaaacgtcgt tctgaactgt ctatcatgat ccaggttaaa
  7201	atcctgcaca ccaccaaatc tccggcggtt taaacttaat taacggcact cctcagccaa
  7261	gtcaaaagcc tccgaccgga ggcttttgac tacatgccca tggcgtttac gccccgccct
  7321	gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac
  7381	aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat
  7441	atttgcccat agtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa
  7501	aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt
  7561	tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa
  7621	actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat
  7681	ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg
  7741	ccatacggaa ctccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat
  7801	aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg
  7861	tctggttata ggtacattTa gcaactgact gaaatgcctc aaaatgttct ttacgatgcc
  7921	attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttcctcag
  7981	ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgaccttatt tcattatggt
  8041	gaaagttgga acctcttacg tgccaAgcca aataggccgt

  (SEQ ID NO: 35)

  1	cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt
  61	tacccacaga ttccgtggat aagcaaaaaa ctaacatgtg aaacaaaaca gcagggccgc
  121	gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt
  181	ttccggtgca tctgtgggaa ccgtgaggct caaccatgaa tctgacagta cgggcgaaac
  241	ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg
  301	ttccgaccct gccgtttacc ggatacctgc tccgcctttc tcccttacgg gaagtgtggc
  361	gctttctcat agcttacaca ctggtatctc ggctcggtgt aggtcgttca ctccaagctg
  421	ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca
  481	cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg
  541	gacttcgcag aggatttgtt tagctaaaca cgccgttgct cttgaagtat gcgccaaagt
  601	ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacaat
  661	taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg
  721	tttacagggc aaaagattac gcgcagaaaa aaaagatctc aagaagatcc tttgatcttt
  781	tctactgaac cactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
  841	agcccaggag gaagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg
  901	cctttcgttt tGCTGAGGag acttagggac cctttaagac tccttattac gcagtatgtt
  961	agcaaacgta gaaaatacat acataaaggt ggcaacatat aaaagaaacg caaagacacc
  1021	gcggaacagg ttgatcttat cgcagtcgat actgaactcg taaggttctc cagcgccaaa
  1081	gacaaaaggg cgacattcaa ccgattgagg gagagaaggt aaatattgac ggaaattatt
  1141	cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgaaaattag agccagcaaa
  1201	atcaccagta acaccattac cattaacaag gccggaaacg tcaccaatga aaccatcgat
  1261	agcagcaccg taatcagtag cgacagaatc aagtttgcct ttagcgtcag actgtagcgc
  1321	gttttcatcg gcattttcgg tcatagcccc cttattagcg tttgccatct tttcataatc
  1381	aaaatcaccg aaaccagagc caccaccgga accgcctccc tcagagccgc caccctcaga
  1441	accgccaccc tcagagccac caccctcaga gccgccacca gaaccaccac cagagccgcc
  1501	gccagcattg acaggaggtt gaggcaggtc agacgattgg ccttgatatt cacaaacgaa
  1561	tggatcctca ttaaagccag aatggaaagc gcagtctctg aatttaccgt tccagtaagc
  1621	gtcatacatg gcttttgatg atacaggagt gtactggtaa taagttttaa cgaggtcagt
  1681	gccttgagta acagtgcccg tataaacagt taatgccccc tgcctatttc ggaacctatt
  1741	attctgaaac atgaaagtat taagaaactg agactcctca aaagaaggat taggattagc
  1801	ggaattttgc tcagtaccag gcggataagt gccgtcgaga gggttgatat aagtatagcc
  1861	cggaataggt gtatcaccgt actcaggagg tttagtaccg ccaccctcag aaccgccacc
  1921	ctcagaaccg ccaccctcag agccaccacc ctcattttca aagatagcaa gcccaataga
  1981	aacccatgta ccgtaacact gagtttcgtc accagtacaa actacaacgc ctgtagcatt
  2041	ccacagacag ccctcatagt tagcgttacg atctaaagtt ttgtcgtctt tccagacgtt
  2101	agtaaatgaa ttttctgtat gggattttgc taaacaactt tcaacagttt cagcggagtg
  2161	agaatagaaa ggaacaacta aaggaattgc gaataataat tttttcattt tttttttcct
  2221	ttactgcacc tgcaggtaat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  2281	ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga
  2341	tttatgggga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca
  2401	ctaacaagtg gcgaatttca tcacggagcc aatgtcctca gcgagtttgt agaaacgcaa
  2461	aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc
  2521	gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat
  2581	ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
  2641	tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac
  2701	cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg gtcaggtggg
  2761	accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct gcgttctgat
  2821	ttaatctgta tcaggctgaa aatcttctct catccgccaa aacagccagg gccctactga
  2881	ctgtttatga caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact
  2941	cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
  3001	aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
  3061	cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga
  3121	aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
  3181	aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
  3241	tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
  3301	gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
  3361	caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg
  3421	caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
  3481	actggtgata ccactcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
  3541	ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt ttcaccaccc
  3601	cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cggtcgataa
  3661	aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
  3721	acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc atactcccac
  3781	cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt
  3841	actggctctt ctcgctaacc caaccggtaa ccccgcttat taaaagcatt ctgtaacaaa
  3901	gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag
  3961	tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt ttatccataa
  4021	gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct ccatacccgt
  4081	ttttttggac gcgtacaact caagtctgac ataaatgacc gctatgagca ctgcaattac
  4141	acgccagatc gttctcGCTa ccGCAaccac cggtatgaac cagattggtg cgcactatga
  4201	aggccacaag atcattgaga ttggtgccgt tgaagtggtg aaccgtcgcc tgacgggcaa
  4261	taacttccat gtttatctca aacccgatcg gctggtggat ccggaagcct ttggcgtaca
  4321	tggtattgcc gatgaatttt tgctcgataa gcccacgttt gccgaagtag ccgatgagtt
  4381	catggactat attcgcggcg cggagttggt gatccataac gcagcgttcg atatcggctt
  4441	tatggactac gagttttcgt tgcttaagcg cgatattccg aagaccaata ctttctgtaa
  4501	ggtcaccgat agccttgcgg tggcgaggaa aatgtttccc ggtaagcgca acagcctcga
  4561	tgcgttatgt gctcgctacg aaatacataa cagtaaacga acgctgcacg gggcattact
  4621	cgatgcccag atccttgcgg aagtttatct ggcgatgacc ggtggtcaaa cgtcgatggc
  4681	ttttgcgatg gaaggagaga cacaacagca acaaggtgaa gcaacaattc agcgcattgt
  4741	acgtcaggca agtaagttac gcgttgtttt tgcgacagat gaagagattg cagctcatga
  4801	agcccgtctc gatctggtgc agaagaaagg cggaagttgc ctctggcgag cataatttaa
  4861	tatcagtaaa ccggacataa cccatgaaga aaaatcgcgc ttttttgaag tgggcagggg
  4921	gcaagtatcc cctgcttgat gatattaaac ggcatttgcc caagggcgaa tgtctggttg
  4981	agccttttgt aggtgccggg tcggtgtttc tcaacaccga cttttctcgt tatatccttg
  5041	ccgatatcaa tagcgacctg atcagtctct ataacattgt gaagatgcgt actgatgagt
  5101	acgtacaggc cgcacgcgag ctgtttgttc ccgaaacaaa ttgcgccgag gtttactatc
  5161	agttccgcga agagttcaac aaaagccagg atccgttccg tcgggcggta ctgtttttat
  5221	atttgaaccg ctacggttac aacggcctgt gtcgttacaa tctgcgcggt gagtttaacg
  5281	tgccgttcgg ccgctacaaa aaaccctatt tcccggaagc agagttgtat cacttcgctg
  5341	aaaaagcgca gaatgccttt ttctattgtg agtcttacgc cgatagcatg gcgcgcgcag
  5401	atgatgcatc cgtcgtctat tgcgatccgc cttatgcacc gctgtctgcg accgccaact
  5461	ttacggcgta tcacacaaac aghtttacgc ttgaacaaca agcgcatctg gcggagatcg
  5521	ccgaaggtct ggttgagcgc catattccag tgctgatctc caatcacgat acgatgttaa
  5581	cgcgtgagtg gtatcagcgc gcaaaattgc atgtcgtcaa agttcgacgc agtataagca
  5641	gcaacggcgg cacacgtaaa aaggtggacg aactgctggc tttgtacaaa ccaggagtcg
  5701	tttcacccgc gaaaaaataa ttcagctaag acactgcact ggattaagat gaaaacgatt
  5761	gaagttgatg atgaactcta cagctatatt gccagccaca ctaagcatat cggcgagagc
  5821	gcatccgaca ttttacggcg tatgttgaaa ttttccgccg catcacagcc tgctgctccg
  5881	gtgacgaaag aggttcgcgt tgcgtcacct gctatcgtcg aagcgaagcc ggtcaaaacg
  5941	attaaagaca aggctcgcgc aatgcgtgaa cttctgcttt cggatgaata cgcagagcaa
  6001	aagcgagcgg tcaatcgctt tatgctgctg ttgtctacac tatattctct tgacgcccag
  6061	gcgtttgccg aagcaacgga atcgttgcac ggtcgtacac gcgtttactt tgcggcagat
  6121	gaacaaacgc tgctgaaaaa tggtaatcag accaagccga aacatgtgcc aggcacgccg
  6181	tattgggtga tcaccaacac caacaccggc cgtaaatgca gcatgatcga acacatcatg
  6241	cagtcgatgc aattcccggc ggaattgatt gagaaggttt gcggaactat ctaacggctg
  6301	aaattaatga ggtcataccc aaatggatag ttcgtttacg cccattgaac aaatgctaaa
  6361	atttcgcgcc agccgccacg aagattttcc ttatcaggag atccttctga ctcgtctttg
  6421	catgcacatg caaagcaagc tgctggagaa ccgcaataaa atgctgaagg ctcagggAat
  6481	taacgagacg ttgtttatgg cgttgattac gctggagtct caggaaaacc acagtattca
  6541	gccttctgaa ttaagttgtg ctcttggatc atcccgtacc aacgcgacgc gtattgccga
  6601	tgaactggaa aaacgcggtt ggatcgaacg tcgtgaaagc gataacgatc gccgctgcct
  6661	gcatctgcaa ttaacggaaa aaggtcacga gtttttgcgc gaggttttac caccgcagca
  6721	taactgcctg catcaactct ggtccgcgct cagcacaaca gaaaaagatc agctcgagca
  6781	aatcacccgc aaattgctct cccgtctcga ccagatggaa caagacggtg tggttctcga
  6841	agcgatgagc taataataca aaaattagga ggaatttcaa catgacaaat ttatctgaca
  6901	tcattgaaaa agaaacagga aaacaactag tgattcaaga atcaattcta atgttaccag
  6961	aagaagtaga ggaagtaatt gggaataaac cagaaagtga tattttagtt catactgctt
  7021	atgatgaaag tacagatgaa aatgtaatgc tattaacttc agatgctcca gaatataaac
  7081	cttgggcttt agtaattcaa gacagtaatg gaaaaaataa aattaaaatg ttataagtcg
  7141	agattaagta aaccggaatc tgaagatgac cgacgcggaa tacgttcgta tccacgaaaa
  7201	actggacatc tacaccttca aaaaacagtt cttcaacaac aaaaaatctg tttctcaccg
  7261	ttgctacgtt ctgttcgaac tgaaacgtcg tggtgaacgt cgtgcgtgct tctggggtta
  7321	cgcggttaac aaaccgcagt ctggtaccga acgtggtatc cacgcggaaa tcttctctat
  7381	ccgtaaagtt gaagaatacc tgcgtgacaa cccgggtcag ttcaccatca actggtactc
  7441	ttcttggtct ccgtgcgcgg actgcgcgga aaaaatcctg gaatggtaca accaggaact
  7501	gcgtggtaac ggtcacaccc tgaaaatctg ggcgtgcaaa ctgtactacg aaaaaaacgc
  7561	gcgtaaccag atcggtctgt ggaacctgcg tgacaacggt gttggtctga acgttatggt
  7621	ttctgaacac taccagtgct gccgtaaaat cttcatccag tcttctcaca accagctgaa
  7681	cgaaaaccgt tggctggaaa aaaccctgaa acgtgcggaa aaacgtcgtt ctgaactgtc
  7741	tatcatgatc caggttaaaa tcctgcacac caccaaatct ccggcggttt aaacttaatt
  7801	aacggcactc ctcagccaag tcaaaagcct ccgaccggag gcttttgact acatgcccat
  7861	ggcgtttacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
  7921	ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
  7981	ttgtcgcctt acgtataata tttgcccata gtgaaaacgg gggcgaagaa gttgtccata
  8041	ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
  8101	atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
  8161	tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
  8221	aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
  8281	agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag gcgggcaaga
  8341	atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
  8401	gtaatatcca gccgaacggt ctggttatag gtacattTag caactgactg aaatgcccca
  8461	aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
  8521	tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt
  8581	gatcttattt cattatggtg aaagttggaa cctcttacgt gccaAgccaa ataggccgt

• DP2

  LOCUSpJC184 6537 bp DNA circular

  FEATURES Location/Qualifiers

  modified_base 852..866

  /note=“USER junction”

  terminator 867..911

  /note=“rrnB1 transcriptional terminator”

  modified_base 919..933

  /note=“USER junction”

  rep_origin complement(39..777)

  /dnas_title=“cloDF13”

  /vntifkey=“33”

  /label=cloDF13

  terminator complement(7817..7852)

  /note=“P14/tonB bidirectional terminator”

  /note=“termination of cat transcript is slightly weaker

  than in opposite direction”

  modified_base 7853..7865

  /note=“USER junction”

  CDS complement(7866..8525)

  /note=“cat (CmR)”

  /note=“from pACYCDuet-1”

  modified_base 7941..7941

  /note=“mutation”

  /note=“annotated as a G in pACYCDuet cat marker

  annotation, here it is an A, but this mutation is silent

  from codon GTC (Val, 25% codon usage) to GTT (Val,

  21% usage), so it should not be of functional relevance”

  promoter 8526..8623

  /note=“cat promoter”

  /note=“from pACYCDuet-1”

  modified_base 8624..8639

  /note=“USER junction”

  CDS complement(2885..3763)

  /dnas_title=“araC”

  /vntifkey=“4”

  /label=araC

  misc_feature 2936..2936

  /note=“originally an ‘a’ in annotation”

  misc_feature 2975..2975

  /note=“originally a t in annotation”

  misc_feature 3041..3041

  /note=“originally an a in annotation”

  misc_feature 3245..3245

  /note=“originally an c in annotation”

  misc_feature 3410..3410

  /note=“originally an a in annotation”

  misc_feature 3569..3569

  /note=“originally a g in annotation”

  misc_feature 3716..3716

  /note=“originally a g in annotation”

  CDS complement(934..2208)

  /dnas_title=“III”

  /vntifkey=“4”

  /label=III

  misc_feature complement(1021..1066)

  /note=“modified to remove internal promoter”

  misc_feature 2423..2435

  /note=“USER linker”

  RBS complement(2209..2222)

  /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.

  1992)”

  modified_base complement(2223..2237)

  /note=“USER junction”

  terminator complement(2443..2867)

  /note=“rrnB1 transcriptional terminator”

  misc_feature 2868..2884

  /note=“USER linker”

  modified_base 789..806

  /note=“USER Junction”

  misc_difference 912..918

  /note=“cloning scar”

  protein_bind complement(2408..2422)

  /note=“UAS II”

  protein_bind complement(2386..2404)

  /note=“UAS I”

  prim_transcript complement(2297..2297)

  /note=“Transcription Start Site”

  enhancer complement(2375..2422)

  /note=“Protected by pspF”

  RBS complement(2406..2415)

  /note=“pspF RBS”

  promoter 2356..2384

  /note=“pspF P1”

  promoter 2375..2402

  /note=“pspF P2”

  promoter 2242..2269

  /note=“pspF P3”

  −35_signal 2242..2247

  /note=“−35”

  −10_signal 2264..2269

  /note=“−10”

  −35_signal 2356..2361

  /note=“−35”

  −35_signal 2375..2380

  /note=“−35”

  −10_signal 2379..2384

  /note=“−10”

  −10_signal 2397..2402

  /note=“−10”

  prim_transcript 2276..2276

  /note=“”

  prim_transcript 2390..2390

  /note=“”

  prim_transcript 2406..2406

  /note=“”

  promoter complement(2308..2324)

  /note=“Sigma54 Core Promoter”

  −35_signal complement(2321..2322)

  /note=“−24”

  −10_signal complement(2309..2310)

  /note=“−12”

  protein_bind complement(2326..2358)

  /note=“High Affinity IHF Site”

  protein_bind complement(2278..2296)

  /note=“tetR binding site”

  misc_feature 3861..3861

  /dnas_title=“C to A ***”

  /vntifkey=“21”

  /label=C to A ***

  misc_feature 3779..3779

  /dnas_title=“A to G ***”

  /vntifkey=“21”

  /label=A to G ***

  prim_transcript complement(3927..3927)

  /note=“pC TSS”

  protein_bind 3950..3966

  /note=“araO1”

  protein_bind 3929..3945

  /note=“araO1”

  protein_bind 3792..3808

  /note=“araO2”

  protein_bind 3971..3992

  /note=“CAP”

  misc_feature 4051..4051

  /note=“”

  misc_feature 4037..4037

  /note=“”

  prim_transcript 4075..4075

  /note=“pBAD TSS”

  protein_bind 4003..4019

  /note=“ara I1”

  protein_bind 4024..4040

  /note=“araI2”

  −10_signal 4061..4066

  /note=“−10”

  −35_signal 4037..4042

  /note=“−35”

  promoter 3971..4100

  /dnas_title=“pBAD”

  /vntifkey=“30”

  /label=pBAD

  RBS 4104..4123

  /note=“Native dnaQ RBS”

  CDS 4124..4855

  /dnas_title=“dnaQ926”

  /vntifkey=“4”

  /label=dnaQ926

  conflict 4157..4159

  /note=“D12A”

  conflict 4163..4165

  /note=“E14A”

  RBS 4865..4883

  /note=“Modified mutS RBS”

  modified_base 4865..4878

  /note=“USER Junction”

  CDS 4884..5720

  /note=“dam (wt)”

  CDS 5749..6294

  /note=“seqA (wt)”

  RBS 5729..5748

  /note=“seqA Native RBS”

  CDS 6323..6853

  /note=“emrR (wt)”

  RBS 6303..6322

  /note=“native emrR RBS”

  modified_base 6275..6292

  /note=“USER Junction”

  CDS 6882..7136

  /note=“PBS2 UGI”

  RBS 6861..6881

  /note=“native UGI RBS”

  modified_base 6858..6877

  /note=“USER Junction”

  RBS 7145..7165

  /note=“dnaE RBS”

  modified_base 7141..7159

  /note=“USER Junction”

  CDS 7166..7792

  /note=“pmCDA1 (opt)”

  modified_base 7793..7809

  /note=“USER junction”

  source 1..5702

• DP3

  LOCUSpJC184 6537 bp DNA circular

  FEATURES Location/Qualifiers

  modified_base 852..866

  /note=“USER junction”

  terminator 867..911

  /note=“rrnB1 transcriptional terminator”

  modified_base 919..933

  /note=“USER junction”

  rep_origin complement(39..777)

  /dnas_title=“cloDF13”

  /vntifkey=“33”

  /label=cloDF13

  terminator complement(5745..5780)

  /note=“P14/tonB bidirectional terminator”

  /note=“termination of cat transcript is slightly weaker

  than in opposite direction”

  modified_base 5781..5793

  /note=“USER junction”

  CDS complement(5794..6453)

  /note=“cat (CmR)”

  /note=“from pACYCDuet-1”

  modified_base 5869..5869

  /note=“mutation”

  /note=“annotated as a G in pACYCDuet cat marker

  annotation, here it is an A, but this mutation is silent

  from codon GTC (Val, 25% codon usage) to GTT (Val,

  21% usage), so it should not be of functional relevance”

  promoter 6454..6551

  /note=“cat promoter”

  /note=“from pACYCDuet-1”

  modified_base 6552..6567

  /note=“USER junction”

  CDS complement(2885..3763)

  /dnas_title=“araC”

  /vntifkey=“4”

  /label=araC

  misc_feature 2936..2936

  /note=“originally an ‘a’ in annotation”

  misc_feature 2975..2975

  /note=“originally a t in annotation”

  misc_feature 3041..3041

  /note=“originally an a in annotation”

  misc_feature 3245..3245

  /note=“originally an c in annotation”

  misc_feature 3410..3410

  /note=“originally an a in annotation”

  misc_feature 3569..3569

  /note=“originally a g in annotation”

  misc_feature 3716..3716

  /note=“originally a g in annotation”

  CDS complement(934..2208)

  /dnas_title=“III”

  /vntifkey=“4”

  /label=III

  misc_feature complement(1021..1066)

  /note=“modified to remove internal promoter”

  misc_feature 2423..2435

  /note=“USER linker”

  RBS complement(2209..2222)

  /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.

  1992)”

  modified_base complement(2223..2237)

  /note=“USER junction”

  terminator complement(2443..2867)

  /note=“rrnB1 transcriptional terminator”

  misc_feature 2868..2884

  /note=“USER linker”

  modified_base 789..806

  /note=“USER Junction”

  misc_difference 912..918

  /note=“cloning scar”

  protein_bind complement(2408..2422)

  /note=“UAS II”

  protein_bind complement(2386..2404)

  /note=“UAS I”

  prim_transcript complement(2297..2297)

  /note=“Transcription Start Site”

  enhancer complement(2375..2422)

  /note=“Protected by pspF”

  RBS complement(2406..2415)

  /note=“pspF RBS”

  promoter 2356..2384

  /note=“pspF P1”

  promoter 2375..2402

  /note=“pspF P2”

  promoter 2242..2269

  /note=“pspF P3”

  −35_signal 2242..2247

  /note=“−35”

  −10_signal 2264..2269

  /note=“−10”

  −35_signal 2356..2361

  /note=“−35”

  −35_signal 2375..2380

  /note=“−35”

  −10_signal 2379..2384

  /note=“−10”

  −10_signal 2397..2402

  /note=“−10”

  prim_transcript 2276..2276

  /note=“”

  prim_transcript 2390..2390

  /note=“”

  prim_transcript 2406..2406

  /note=“”

  promoter complement(2308..2324)

  /note=“Sigma54 Core Promoter”

  −35_signal complement(2321..2322)

  /note=“−24”

  −10_signal complement(2309..2310)

  /note=“−12”

  protein_bind complement(2326..2358)

  /note=“High Affinity IHF Site”

  protein_bind complement(2278..2296)

  /note=“tetR binding site”

  misc_feature 3861..3861

  /dnas_title=“C to A ***”

  /vntifkey=“21”

  /label=C to A ***

  misc_feature 3779..3779

  /dnas_title=“A to G ***”

  /vntifkey=“21”

  /label=A to G ***

  prim_transcript complement(3927..3927)

  /note=“pC TSS”

  protein_bind 3950..3966

  /note=“araO1”

  protein_bind 3929..3945

  /note=“araO1”

  protein_bind 3792..3808

  /note=“araO2”

  protein_bind 3971..3992

  /note=“CAP”

  misc_feature 4051..4051

  /note=“”

  misc_feature 4037..4037

  /note=“”

  prim_transcript 4075..4075

  /note=“pBAD TSS”

  protein_bind 4003..4019

  /note=“ara I1”

  protein_bind 4024..4040

  /note=“araI2”

  −10_signal 4061..4066

  /note=“−10”

  −35_signal 4037..4042

  /note=“−35”

  promoter 3971..4100

  /dnas_title=“pBAD”

  /vntifkey=“30”

  /label=pBAD

  RBS 4104..4123

  /note=“Native dnaQ RBS”

  CDS 4124..4855

  /dnas_title=“dnaQ926”

  /vntifkey=“4”

  /label=dnaQ926

  conflict 4157..4159

  /note=“D12A”

  conflict 4163..4165

  /note=“E14A”

  RBS 4865..4883

  /note=“Modified mutS RBS”

  modified_base 4865..4878

  /note=“USER Junction”

  CDS 4884..5720

  /note=“dam (wt)”

  modified_base 5721..5737

  /note=“USER junction”

  source 1..6567

• DP4

  LOCUSpJC184 6537 bp DNA circular

  FEATURES Location/Qualifiers

  modified_base 852..866

  /note=“USER junction”

  terminator 867..911

  /note=“rrnB1 transcriptional terminator”

  modified_base 919..933

  /note=“USER junction”

  rep_origin complement(39..777)

  /dnas_title=“cloDF13”

  /vntifkey=“33”

  /label=cloDF13

  terminator complement(6319..6354)

  /note=“P14/tonB bidirectional terminator”

  /note=“termination of cat transcript is slightly weaker

  than in opposite direction”

  modified_base 6355..6367

  /note=“USER junction”

  CDS complement(6368..7027)

  /note=“cat (CmR)”

  /note=“from pACYCDuet-1”

  modified_base 6443..6443

  /note=“mutation”

  /note=“annotated as a G in pACYCDuet cat marker

  annotation, here it is an A, but this mutation is silent

  from codon GTC (Val, 25% codon usage) to GTT (Val,

  21% usage), so it should not be of functional relevance”

  promoter 7028..7125

  /note=“cat promoter”

  /note=“from pACYCDuet-1”

  modified_base 7126..7141

  /note=“USER junction”

  CDS complement(2885..3763)

  /dnas_title=“araC”

  /vntifkey=“4”

  /label=araC

  misc_feature 2936..2936

  /note=“originally an ‘a’ in annotation”

  misc_feature 2975..2975

  /note=“originally a t in annotation”

  misc_feature 3041..3041

  /note=“originally an a in annotation”

  misc_feature 3245..3245

  /note=“originally an c in annotation”

  misc_feature 3410..3410

  /note=“originally an a in annotation”

  misc_feature 3569..3569

  /note=“originally a g in annotation”

  misc_feature 3716..3716

  /note=“originally a g in annotation”

  CDS complement(934..2208)

  /dnas_title=“III”

  /vntifkey=“4”

  /label=III

  misc_feature complement(1021..1066)

  /note=“modified to remove internal promoter”

  misc_feature 2423..2435

  /note=“USER linker”

  RBS complement(2209..2222)

  /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.

  1992)”

  modified_base complement(2223..2237)

  /note=“USER junction”

  terminator complement(2443..2867)

  /note=“rrnB1 transcriptional terminator”

  misc_feature 2868..2884

  /note=“USER linker”

  modified_base 789..806

  /note=“USER Junction”

  misc_difference 912..918

  /note=“cloning scar”

  protein_bind complement(2408..2422)

  /note=“UAS II”

  protein_bind complement(2386..2404)

  /note=“UAS I”

  prim_transcript complement(2297..2297)

  /note=“Transcription Start Site”

  enhancer complement(2375..2422)

  /note=“Protected by pspF”

  RBS complement(2406..2415)

  /note=“pspF RBS”

  promoter 2356..2384

  /note=“pspF P1”

  promoter 2375..2402

  /note=“pspF P2”

  promoter 2242..2269

  /note=“pspF P3”

  −35_signal 2242..2247

  /note=“−35”

  −10_signal 2264..2269

  /note=“−10”

  −35_signal 2356..2361

  /note=“−35”

  −35_signal 2375..2380

  /note=“−35”

  −10_signal 2379..2384

  /note=“−10”

  −10_signal 2397..2402

  /note=“−10”

  prim_transcript 2276..2276

  /note=“”

  prim_transcript 2390..2390

  /note=“”

  prim_transcript 2406..2406

  /note=“”

  promoter complement(2308..2324)

  /note=“Sigma54 Core Promoter”

  −35_signal complement(2321..2322)

  /note=“−24”

  −10_signal complement(2309..2310)

  /note=“−12”

  protein_bind complement(2326..2358)

  /note=“High Affinity IHF Site”

  protein_bind complement(2278..2296)

  /note=“tetR binding site”

  misc_feature 3861..3861

  /dnas_title=“C to A ***”

  /vntifkey=“21”

  /label=C to A ***

  misc_feature 3779..3779

  /dnas_title=“A to G ***”

  /vntifkey=“21”

  /label=A to G ***

  prim_transcript complement(3927..3927)

  /note=“pC TSS”

  protein_bind 3950..3966

  /note=“araO1”

  protein_bind 3929..3945

  /note=“araO1”

  protein_bind 3792..3808

  /note=“araO2”

  protein_bind 3971..3992

  /note=“CAP”

  misc_feature 4051..4051

  /note=“”

  misc_feature 4037..4037

  /note=“”

  prim_transcript 4075..4075

  /note=“pBAD TSS”

  protein_bind 4003..4019

  /note=“ara I1”

  protein_bind 4024..4040

  /note=“araI2”

  −10_signal 4061..4066

  /note=“−10”

  −35_signal 4037..4042

  /note=“−35”

  promoter 3971..4100

  /dnas_title=“pBAD”

  /vntifkey=“30”

  /label=pBAD

  RBS 4104..4123

  /note=“Native dnaQ RBS”

  CDS 4124..4855

  /dnas_title=“dnaQ926”

  /vntifkey=“4”

  /label=dnaQ926

  conflict 4157..4159

  /note=“D12A”

  conflict 4163..4165

  /note=“E14A”

  RBS 4865..4883

  /note=“Modified mutS RBS”

  modified_base 4865..4878

  /note=“USER Junction”

  CDS 4884..5720

  /note=“dam (wt)”

  CDS 5749..6294

  /note=“seqA (wt)”

  RBS 5729..5748

  /note=“seqA Native RBS”

  modified_base 6275..6311

  /note=“USER junction”

  source 1..7141

• DP5

  LOCUSpJC184 6537 bp DNA circular

  FEATURES Location/Qualifiers

  modified_base 852..866

  /note=“USER junction”

  terminator 867..911

  /note=“rrnB1 transcriptional terminator”

  modified_base 919..933

  /note=“USER junction”

  rep_origin complement(39..777)

  /dnas_title=“cloDF13”

  /vntifkey=“33”

  /label=cloDF13

  terminator complement(7258..7293)

  /note=“P14/tonB bidirectional terminator”

  /note=“termination of cat transcript is slightly weaker

  than in opposite direction”

  modified_base 7294..7306

  /note=“USER junction”

  CDS complement(7307..7966)

  /note=“cat (CmR)”

  /note=“from pACYCDuet-1”

  modified_base 7382..7382

  /note=“mutation”

  /note=“annotated as a G in pACYCDuet cat marker

  annotation, here it is an A, but this mutation is silent

  from codon GTC (Val, 25% codon usage) to GTT (Val,

  21% usage), so it should not be of functional relevance”

  promoter 7967..8064

  /note=“cat promoter”

  /note=“from pACYCDuet-1”

  modified_base 8065..8080

  /note=“USER junction”

  CDS complement(2885..3763)

  /dnas_title=“araC”

  /vntifkey=“4”

  /label=araC

  misc_feature 2936..2936

  /note=“originally an ‘a’ in annotation”

  misc_feature 2975..2975

  /note=“originally a t in annotation”

  misc_feature 3041..3041

  /note=“originally an a in annotation”

  misc_feature 3245..3245

  /note=“originally an c in annotation”

  misc_feature 3410..3410

  /note=“originally an a in annotation”

  misc_feature 3569..3569

  /note=“originally a g in annotation”

  misc_feature 3716..3716

  /note=“originally a g in annotation”

  CDS complement(934..2208)

  /dnas_title=“III”

  /vntifkey=“4”

  /label=III

  misc_feature complement(1021..1066)

  /note=“modified to remove internal promoter”

  misc_feature 2423..2435

  /note=“USER linker”

  RBS complement(2209..2222)

  /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.

  1992)”

  modified_base complement(2223..2237)

  /note=“USER junction”

  terminator complement(2443..2867)

  /note=“rrnB1 transcriptional terminator”

  misc_feature 2868..2884

  /note=“USER linker”

  modified_base 789..806

  /note=“USER Junction”

  misc_difference 912..918

  /note=“cloning scar”

  protein_bind complement(2408..2422)

  /note=“UAS II”

  protein_bind complement(2386..2404)

  /note=“UAS I”

  prim_transcript complement(2297..2297)

  /note=“Transcription Start Site”

  enhancer complement(2375..2422)

  /note=“Protected by pspF”

  RBS complement(2406..2415)

  /note=“pspF RBS”

  promoter 2356..2384

  /note=“pspF P1”

  promoter 2375..2402

  /note=“pspF P2”

  promoter 2242..2269

  /note=“pspF P3”

  −35_signal 2242..2247

  /note=“−35”

  −10_signal 2264..2269

  /note=“−10”

  −35_signal 2356..2361

  /note=“−35”

  −35_signal 2375..2380

  /note=“−35”

  −10_signal 2379..2384

  /note=“−10”

  −10_signal 2397..2402

  /note=“−10”

  prim_transcript 2276..2276

  /note=“”

  prim_transcript 2390..2390

  /note=“”

  prim_transcript 2406..2406

  /note=“”

  promoter complement(2308..2324)

  /note=“Sigma54 Core Promoter”

  −35_signal_ complement(2321..2322)

  /note=“−24”

  −10_signal complement(2309..2310)

  /note=“−12”

  protein_bind complement(2326..2358)

  /note=“High Affinity IHF Site”

  protein_bind complement(2278..2296)

  /note=“tetR binding site”

  misc_feature 3861..3861

  /dnas_title=“C to A ***”

  /vntifkey=“21”

  /label=C to A ***

  misc_feature 3779..3779

  /dnas_title=“A to G ***”

  /vntifkey=“21”

  /label=A to G ***

  prim_transcript complement(3927..3927)

  /note=“pC TSS”

  protein_bind 3950..3966

  /note=“araO1”

  protein_bind 3929..3945

  /note=“araO1”

  protein_bind 3792..3808

  /note=“araO2”

  protein_bind 3971..3992

  /note=“CAP”

  misc_feature 4051..4051

  /note=“”

  misc_feature 4037..4037

  /note=“”

  prim_transcript 4075..4075

  /note=“pBAD TSS”

  protein_bind 4003..4019

  /note=“ara I1”

  protein_bind 4024..4040

  /note=“araI2”

  −10_signal 4061..4066

  /note=“−10”

  −35_signal 4037..4042

  /note=“−35”

  promoter 3971..4100

  /dnas_title=“pBAD”

  /vntifkey=“30”

  /label=pBAD

  RBS 4104..4123

  /note=“Native dnaQ RBS”

  CDS 4124..4855

  /dnas_title=“dnaQ926”

  /vntifkey=“4”

  /label=dnaQ926

  conflict 4157..4159

  /note=“D12A”

  conflict 4163..4165

  /note=“E14A”

  RBS 4865..4883

  /note=“Modified mutS RBS”

  modified_base 4865..4878

  /note=“USER Junction”

  CDS 4884..5720

  /note=“dam (wt)”

  CDS 5749..6294

  /note=“seqA (wt)”

  RBS 5729..5748

  /note=“seqA Native RBS”

  modified_base 6275..6292

  /note=“USER Junction”

  CDS 6323..6577

  /note=“PBS2 UGI”

  RBS 6302..6322

  /note=“Native UGI RBS”

  modified_base 6299..6318

  /note=“USER Junction”

  RBS 6586..6606

  /note=“dnaE RBS”

  modified_base 6582..6600

  /note=“USER Junction”

  CDS 6607..7233

  /note=“pmCDA1 (opt)”

  modified_base 7234..7250

  /note=“USER junction”

  source 1..8080

• DP6

  LOCUSpJC184 6537 bp DNA circular

  FEATURES Location/Qualifiers

  modified_base 852..866

  /note=“USER junction”

  terminator 867..911

  /note=“rrnB1 transcriptional terminator”

  modified_base 919..933

  /note=“USER junction”

  rep_origin complement(39..777)

  /dnas_title=“cloDF13”

  /vntifkey=“33”

  /label=cloDF13

  terminator complement(7817..7852)

  /note=“P14/tonB bidirectional terminator”

  /note=“termination of cat transcript is slightly weaker

  than in opposite direction”

  modified_base 7853:7865

  /note=“USER junction”

  CDS complement(7866..8525)

  /note=“cat (CmR)”

  /note=“from pACYCDuet-1”

  modified_base 7941..7941

  /note=“mutation”

  /note=“annotated as a G in pACYCDuet cat marker

  annotation, here it is an A, but this mutation is silent

  from codon GTC (Val, 25% codon usage) to GTT (Val,

  21% usage), so it should not be of functional relevance”

  promoter 8526..8623

  /note=“cat promoter”

  /note=“from pACYCDuet-1”

  modified_base 8624..8639

  /note=“USER junction”

  CDS complement(2885..3763)

  /dnas_title=“araC”

  /vntifkey=“4”

  /label=araC

  misc_feature 2936..2936

  /note=“originally an ‘a’ in annotation”

  misc_feature 2975..2975

  /note=“originally a t in annotation”

  misc_feature 3041..3041

  /note=“originally an a in annotation”

  misc_feature 3245..3245

  /note=“originally an c in annotation”

  misc_feature 3410..3410

  /note=“originally an a in annotation”

  misc_feature 3569..3569

  /note=“originally a g in annotation”

  misc_feature 3716..3716

  /note=“originally a g in annotation”

  CDS complement(934..2208)

  /dnas_title=“III”

  /vntifkey=“4”

  /label=III

  misc_feature complement(1021.. 1066)

  /note=“modified to remove internal promoter”

  misc_feature 2423..2435

  /note=“USER linker”

  RBS complement(2209..2222)

  /note=“sd8 RBS (from Ringquist and Gold Mol. Micro.

  1992)”

  modified_base complement(2223..2237)

  /note=“USER junction”

  terminator complement(2443..2867)

  /note=“rrnBI transcriptional terminator”

  misc_feature 2868..2884

  /note=“USER linker”

  modified_base 789..806

  /note=“USER Junction”

  misc_difference 912..918

  /note=“cloning scar”

  protein_bind complement(2408..2422)

  /note=“UAS II”

  protein_bind complement(2386..2404)

  /note=“UAS I”

  prim_transcript complement(2297..2297)

  /note=“Transeription Start Site”

  enhancer complement(2375..2422)

  /note=“Protected by pspF”

  RBS complement(2406..2415)

  /note=“pspF RBS”

  promoter 2356..2384

  /note=“pspF P1”

  promoter 2375..2402

  /note=“pspF P2”

  promoter 2242..2269

  /note=“pspF P3”

  −35_signal 2242..2247

  /note=“−3”

  −10_signal 2264..2269

  /note=“−10”

  −35_signal 2356..2361

  /note=“−35”

  −35_signal 2375..2380

  /note=“−35”

  −10_signal 2379..2384

  /note=“−10”

  −10_signal 2197..2402

  /note=“−10”

  prim_transcript 2276..2276

  /note=“”

  prim_transcript 2390..2390

  /note=“”

  prim_transcript 2406..2406

  /note=“”

  promoter complement(2308..2324)

  /note=“Sigma54 Core Promoter”

  −35_signal_ complement(2321..2322)

  /note=“−24”

  −10_signal complement(2309..2310)

  /note=“−12”

  protein_bind complement(2326..2358)

  /note=“High Affinity IHF Site”

  protein_bind complement(2278..2296)

  /note=“tetR binding site”

  misc_feature 3861..3861

  /dnas_title=“C to A ***”

  /vntifkey=“21”

  /label=C to A ***

  misc_feature 3779..3779

  /dnas_title=“A to G ***”

  /vntifkey=“21”

  /label=A to G ***

  prim_transcript complement(3927..3927)

  /note=“pC TSS”

  protein_bind 3950..3966

  /note=“araO1”

  protein_bind 3929..3945

  /note=“araO1”

  protein_bind 3792..3808

  /note=“araO2”

  protein_bind 3971..3992

  /note=“CAP”

  misc_feature 4051..4051

  /note=“”

  misc_feature 4037..4037

  /note=“”

  prim_transcript 4075..4075

  /note=“pBAD TSS”

  protein_bind 4003..4019

  /note=“ara I1”

  protein_bind 4024..4040

  /note=“araI2”

  −10_signal 4061..4066

  /note=“−10”

  −35_signal 4037..4042

  /note=“−35”

  promoter 3971..4100

  /dnas_title=“pBAD”

  /vntifkey=“30”

  /label=pBAD

  RBS 4104..4123

  /note=“Native dnaQ RBS”

  CDS 4124..4855

  /dnas_title=“dnaQ926”

  /vntifkey=“4”

  /label=dnaQ926

  conflict 4157..4159

  /note=“D12A”

  conflict 4163..4165

  /note=“E14A”

  RBS 4865..4883

  /note=“Modified mutS RBS”

  modified_base 4865..4878

  /note=“USER Junction”

  CDS 4884..5720

  /note=“−35”

  CDS 5749..6294

  /note=“seqA (wt)”

  RBS 5729..5748

  /note=“seqA Native RBS”

  CDS 6323..6853

  /note=“emrR (wt)”

  RBS 6303..6322

  /note=“native emrR RBS”

  modified_base 6275..6292

  /note=“USER Junction”

  CDS 6882..7136

  /note=“PBS2 UGI”

  RBS 6861..6881

  /note=“Native UGI RBS”

  modified_base 6858..6877

  /note=“USER Junction”

  RBS 7145..7165

  /note=“dnaE RBS”

  modified_base 7141..7159

  /note=“USER Junction”

  CDS 7166..7792

  /note=“pmCDA1 (opt)”

  modified_base 7793..7809

  /note=“USER junction”

  source 1..8639

Exemplary Sequences
• Non-limiting sequences of exemplary encoding gene products that increase the mutation rate in a host cell, e.g., in a bacterial host cell, are provided below. Those of ordinary skill in the art will understand that other useful sequences for each of the gene products provided exist, e.g., sequences having one or more point mutations that do not affect the sequence or function of the encoded RNA or protein.

• araC
  (SEQ ID NO: 33)
  atga caacttgacg gctacatcat tcactttttc ttcacaacca gcacggaact
  cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa
  aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg
  cctggctgat acgttgatcc tcgcgccagc ttaagacgct aatccctaac tgctaggga
  aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa
  aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc cgattatcca
  tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca
  gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta atgatttgcc
  caaacaggtc gctgaaatgc ggctgatgcg cr tcatccg gcgaaagaac cccgtattgg
  caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga aagtaaaccc
  actgatgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc tctcctggcg
  ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccccgattt ttcaccaccc
  cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt cgatcgataa
  aaaaatcgag ataaccgtta gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa
  acgagtatcc cggcagcagg ggatcatttt gcgcttcagc cat
  dnaQ926
  (SEQ ID NO: 37)
  atgag cactgcaatt acacgccaga tcgttctcGC TaccGCAacc accggtatga
  accagattgg tgcgcactat gaaggccaca agatcattga gattggtgcc gttgaagtgg
  tgaaccgtcg cctgacgggc aataacttcc atgtttatct caaacccgat cggctgatgg
  atccggaagc ctttggcgta catggtattg ccgatgaatt tttgctcgat aagcccacgt
  ttgccgaagt agccgatgag ttcatggact atattcgcgg cgcggagttg gtgatccata
  acgcagcatt cgatatcggc tttatggact acgagttttc gttgcttaag cgcgatattc
  cgaaaaccaa tactttctgt aagatcaccg atagccttgc ggtggcgagg aaaatgtttc
  ccggtaagcg caacagcctc gatgcgttat gtgctcgctd cgaaatagat aacagtaaac
  gaacgctgca cggggcatta ctcgatgccc agatccttgc ggaagtttat ctggcgatga
  ccggtagtca aacgtcgatg gcttttgcga tggaaagaga gacacaacag caacaaggtg
  aagcaacaat tcagcgcatt gtacgtcagg caagtaagtt acgcgttgtt tttgcgacag
  atgaagagat tgcagctcat gaagcccgtc tcgatctggt gcagagaaa ggcggaagtt
  gcctctggcg agcataa
  dam
  (SEQ ID NO: 38)
  atgaa gaaaaatcgc
  gcttttttga agtgggcagg gggcaagtat cccctgcttg atgatattaa acggcatttg
  cccaagggcg aatgtctggt tgagcctttt gtaggtgccg gatcggtgtt tctcaacacc
  gacttttctc gttatatcct tgccgatatc aatagcgacc tgatcagtct ctataacatt
  gtgaagatgc gtactgatga gtacgtacag gccgcacgcg agctgtttgt tcccgaaaca
  aattgcgccg aggtttacta tcagttccgc gaagagttca acaaaagcca ggatccgttc
  cgtcgggcgg tactgttttt atatttgaac cgctacaatt acaacggcct gtgtcgttac
  aatctgcgcg gtgagtttaa cgtgccgttc ggccactaca aaaaacccta tttcccggaa
  gcagagttgt atcacttcgc tgaaaaagcg cagaatgcct ttttctattg tgagtcttac
  gccgatagca tggcgcgcgc agatgatgca tccgtcgtct attgcgatcc gccttatgca
  ccgctgtctg cgaccgccaa ctttacggcg tatcacacaa acagttttac acttgaacaa
  caagcgcatc tggcggagat cgccgaaggt ctgattgagc gccatattcc agtgctgatc
  tccaatcacg atacgatgtt aacgcgtgag tggtatcagc gcgcaaaatt gcatatcgtc
  aaagttcgac gcagtataaa cagcaacggc ggcacacgta aaaaggtgga cgaactgctg
  gctttgtaca aaccaggagt cgtttcaccc gcgaaaaaat aa
  seqA
  (SEQ ID NO: 39)
  atgaaaacga ttaaagttga tgatgaactc tacagctata ttaccagcca
  cactaagcat atcggcgaga gcgcatccga cattttacgg cgtatgttga aattttccgc
  cgcatcacag cctgctgctc cggtgacgaa agaggttcgc gttgcgtcac ctgctatcgt
  cgaagcgaag ccggtcaaaa cgattaaaga caaggttcgc gcaatgcgtg aacttctgct
  ttcggatgaa tacgcagagc aaaagcgagc ggtcaatcgc tttatgctgc tgttgtctac
  actatattct cttgacgccc aggcgtttgc cgaagcaacg gaatcgttgc acggtcgtac
  acgcgtttac tttgcggcag atgaacaaac gctgctgaaa aatagtaatc agaccaagcc
  gaaacatgtg ccaggcacgc cgtattaggt gatcaccaac accaacaccg gccgtaaatg
  cagcatgatc gaacacatca tgcagtcgat gcaattcccg gcggaattga ttgagaaggt
  ttgcggaact atctaa
  emrR
  (SEQ ID NO: 40)
  atggat agttcgttta
  cgcccattga acaaatgcta aaatttcgcg ccagccgcca caaagacttt ccttatcagg
  agatccttct gactcgtctt tgcatgcaca tgcaaagcaa gctgctggag aaccgcaata
  aaatactgaa ggctcagggA attaacgaga cgttatttat ggcgttgatt acgctggagt
  ctcaggaaaa ccacagtatt cagccttctg aattaagttg tgctcttgga tcatcccgta
  ccaacgcgac gcgtattgcc gatgaactgg aaaaacgcgg ttggatcgaa cgtcgtgaaa
  gcgataacga tcgccgctgc ctgcatctgc aattaacgga aaaaggtcac aagtttttgc
  gcgaggtttt accaccgcag cataactgcc tgcatcaact ctggtccgcg ctcagcacaa
  cagaaaaaga tcagctcgag caaatcaccc gcaaattgct ctcccgtctc gaccagatgg
  aacaagacgg tgtggttctc gaagcgatga gctaa
  UGI
  (SEQ ID NO: 41)
  atgacaa atttatctga catcattgaa aaagaaacag gaaaattaact agtgattcaa
  gaatcaattc taatgttacc agaagaagta gaggaagtaa ttgggaataa accagaaagt
  gatattttag ttcatactgc ttatgatgaa agtacagatg aaaatgtaat gctattaact
  tcagatgctc cagaatataa accttgggct ttagtaattc aagacagtaa tggagaaaat
  aaaattaaaa tgttataa
  CDA1
  (SEQ ID NO: 42)
  atg accgacgcgg
  aatacgttcg tatccacgaa aaactggaca tctacacctt caaaaaacag ttcttcaaca
  acaaaaaatc tctttctcac cgttgctacg ttctgttcga actgaaacgt cgtgatgaac
  gtcgtgcgtg cttctgaggt tacgcgatta acaaaccgca gtctgatacc gaacgtggta
  tccacgcgga aatcttctct atccgtaaag ttgaagaata cctgcgtgac aacccgggtc
  agttcaccat caactggtac tcttcttaat ctccgtgcgc gaactgcgcg gaaaaaatcc
  tggaatggta caaccaggaa ctgcgtggta acggtcacac cctgaaaatc tgggcgtgca
  aactgtacta cgaaaaaaac gcgcgtaacc agatcggtct gtggaacctg cgtgacaacg
  gtattggtct gaacgttatg gtttctgaac actaccagtg ctgccgtaaa atcttcatcc
  agtcttctca caaccagctg aacgaaaacc gttggctgga aaaaaccctg aaacgtgcgg
  aaaaacgtcg ttctgaacta tctatcatga tccaggttaa aatcctgcac accaccaaat
  ctccggcggt ttaa
  Drift Promoter
  (SEQ ID NO: 124)
  aat gttgtcctct tgatttctgc gttcaggatt gtcctgctct
  ctatcactga tagggatgaa ctgttaatac aatttgcgtg ccaatttttt atctttttga
  tttataaaga tctgattgaa gaatcaacag caacatgcca ggatgagtta gcgaattaca
  ctaacaagtg gcgaatttca tc

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• All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.
EQUIVALENTS AND SCOPE
• Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.
• Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
• It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
• Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
• Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
• In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Claims (21)

1-125. (canceled)

126. An expression construct for modulating the mutation rate of nucleic acids in a bacterial cell, the construct comprising:

(i) a drift promoter; and

(ii) a nucleic acid sequence encoding one or more gene products selected from a gene product that disrupts a proofreading pathway, a translesion synthesis pathway, a methyl-directed mismatch repair pathway, a base excision repair pathway, or a base selection pathway of the bacterial cell, or any combination thereof,

wherein the nucleic acid sequence encoding the one or more gene products is under the control of the drift promoter.

127. The expression construct ofclaim 126, wherein the gene product that disrupts a proofreading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product.

128. The expression construct ofclaim 126, wherein the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product.

129. The expression construct ofclaim 126, wherein the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product.

130. The expression construct ofclaim 126, wherein the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product.

131. The expression construct ofclaim 126, wherein the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product.

132. The expression construct ofclaim 126, wherein the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product.

133. The expression construct ofclaim 126, wherein the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product.

134. The expression construct ofclaim 126, wherein the expression construct comprises a nucleic acid sequence encoding a dnaE74, a dnaE486, a dnaE1026, a dnaX36, a dnaX2016, a rpsD12, a rpsD14, a rpsD16, a polB, a polB(D156A), a MAG1, a AAG(Y127I-H136L), and/or a Δ80-AAG(Y127I-H136L) gene product.

135. The expression construct ofclaim 126, wherein the one or more gene product is dnaQ926, umuD′, umuC, recA730, dam, seqA, emrR, PBS2, UGI, or CDA1, or any combination thereof.

136. The expression construct ofclaim 126, wherein the expression construct comprises a nucleic acid sequence encoding a: dnaQ926 gene product, a dam gene product, a seqA gene product, an emrR gene product, a ugi gene product, and a CDA1 gene product.

137. The expression construct ofclaim 126 comprising the sequence set forth in any one of SEQ ID NO: 27, 28, 29, 33, 34, and 35.

138. The expression construct ofclaim 126, wherein the drift promoter comprises the sequence set forth in SEQ ID NO: 124.

139. The expression construct ofclaim 126, wherein the expression construct comprises a nucleic acid sequence encoding an arabinose operon regulatory protein.

140. The expression construct ofclaim 139, wherein the nucleic acid sequence encoding an arabinose operon regulatory protein comprises the sequence set forth in SEQ ID NO: 36.

141. A cell comprising the expression construct ofclaim 126.

142. A plasmid comprising the expression construct ofclaim 126.

143. The plasmid ofclaim 142, wherein the plasmid comprises a bacterial origin of replication.

144. The plasmid ofclaim 143, wherein the origin of replication is a cloDF13 origin of replication.

145. The plasmid ofclaim 142 further comprising an expression construct comprising a nucleic acid sequence encoding pIII protein.

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