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.2019 Apr 18;19(8):1846.
doi: 10.3390/s19081846.

A Biosensor Platform for Metal Detection Based on Enhanced Green Fluorescent Protein

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A Biosensor Platform for Metal Detection Based on Enhanced Green Fluorescent Protein

Woonwoo Lee et al. Sensors (Basel)..

Abstract

Microbial cell-based biosensors, which mostly rely on stress-responsive operons, have been widely developed to monitor environmental pollutants. Biosensors are usually more convenient and inexpensive than traditional instrumental analyses of environmental pollutants. However, the targets of biosensors are restricted by the limited number of genetic operon systems available. In this study, we demonstrated a novel strategy to overcome this limitation by engineering an enhanced green fluorescent protein (eGFP). It has been reported that combining two fragments of split-eGFP can form a native structure. Thus, we engineered new biosensors by inserting metal-binding loops (MBLs) between β-strands 9 and 10 of the eGFP, which then undergoes conformational changes upon interaction between the MBLs and targets, thereby emitting fluorescence. The two designed MLBs based on our previous study were employed as linkers between two fragments of eGFP. As a result, anEscherichia coli biosensor exhibited a fluorescent signal only when interacting with cadmium ions, revealing the prospect of a new biosensor for cadmium detection. Although this study is a starting stage for further developing biosensors, we believe that the proposed strategy can serve as basis to develop new biosensors to target various environmental pollutants.

Keywords: biosensors; eGFP; heavy metal; split-protein systems.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Diagram of mechanism for engineered eGFP as sensing molecule. The eGFP is active in its native form, whereas the engineered eGFP is inactive due to loop insertion. When a metal ion binds to the MBL, the two parts of the engineered eGFP approach each other and associate to become active. (Red lines, MBL inserted into the eGFP; black dots, metal ions).
Figure 2
Figure 2
Induction coefficients fromE. coli cells harboring eGFP-loop2 exposed to metal ions. (a) Responses of engineered eGFP-loop2 to various metal(loid) ions. (b) Induction coefficients of eGFP-loop2 to different concentrations of Cd, Ni, and Hg. (Ctl, control without metal ions).
Figure 3
Figure 3
Concentration-dependent responses ofE. coli cells with eGFP-loop 2 to Cd ions. (a) Induction coefficients for Cd concentrations from 0 to 5 µM. (b) Linear regression of Cd induction coefficient according to concentration. (y, induction coefficient;x, concentration of Cd;R2, coefficient of determination from linear regression).
Figure 3
Figure 3
Concentration-dependent responses ofE. coli cells with eGFP-loop 2 to Cd ions. (a) Induction coefficients for Cd concentrations from 0 to 5 µM. (b) Linear regression of Cd induction coefficient according to concentration. (y, induction coefficient;x, concentration of Cd;R2, coefficient of determination from linear regression).
Figure 4
Figure 4
Structures of X-ray crystallographic eGFP and model structures with modifications. (a) Wild type structure of eGFP (4ka9.pdb). (b) Model structure of eGFP-loop1 (CNHEPGTVCPIC). (c) Model structure of eGFP-loop2 (CPGDDSADC). (d) Superimposed model of the three structures (modified loops and chromophore are enclosed in red circles).
Figure 5
Figure 5
Ramachandran plots for model structures. (a) Wild type structure of eGFP. (b) Structure of eGFP-loop1. (c) Structure of eGFP-loop2. (The three residues, 65T, 66Y, and 67G, in the chromophore are indicated with red arrows).
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