Construction And Optimization Strategy Of Genetic Circuit Mercury Ion Whole-cell Biosensor

Heavy metal ions are widely used in various production activities and invade the natural environment with wastes,which poses a serious threat to the ecosystem and human health.Among them,heavy metal ion mercury(Hg²⁺)is the most significant harm.Therefore,it’s particularly important for rapid detection and quantification of bioavailable mercury.Whole cell biosensor is a biosynthesis and detection system based on microbial cells.It aims to convert external stimuli into measurable cell responses through internally constructed gene circuits,and realize rapid and simple detection of small molecule effectors by measuring relatively easy to quantify output signals.The sensor has unique advantages such as high selectivity,renewable,easy preparation,low cost,and can detect bioavailable mercury,so it has obvious advantages in the aspect of heavy metal ions in environmental pollutants.Based on the concept of synthetic biology,more and more gene circuit type whole cell biosensors have been designed and realized the specific detection of heavy metal ions Hg²⁺.These sensors are limited by sensitivity,detection limits,and other performance,which cannot meet the needs of actual sample detection.Aimed at these problems,a cascade of multilayer transcriptional signal amplifiers,promoter engineering,the introduction of protein degradation tags,and additional ligand binding sites have been used to modify the sensor performance to reduce background expression in previously reported studies.However,the above optimization strategy construction system is complicated,more simplified signal amplification strategies should be sought to improve the sensitivity of sensors.In this study,the transcriptional regulation mechanism of MerR protein was utilized to construct the whole cell biosensor for the detection of Hg²⁺.In this biosensor,the metalloprotein MerR is used as an induction module for recognizing signals.The homologous bidirectional promoter Pmer is used as a signal processing module for regulating transcription of downstream genes and MerR itself.The stable green fluorescent protein SFGFP is used as the reporter molecule to indicate mercury ion concentration.On the basis of the design of this gene loop,the following four relatively simple modification strategies are tried to change the performance of the sensor,and the influence of these strategies on the sensor sensitivity,detection limit,background expression,dynamic output range,and other performance is systematically explored.Through the above optimization strategies,we expect to obtain excellent sensors in all aspects for the detection of trace mercury contaminated samples with high selectivity and high sensitivity.The specific research contents and conclusions of this study are as follows:（1）Study the influence of copy number of expression vector on sensor performance:Two kinds of whole-cell biosensors using different copy number plasmids as expression vectors were constructed by molecular cloning technology.Compared with the sensor with low copy plasmids,the detection limit of high copy plasmids was1.6n M,and the sensitivity of the sensor to Hg²⁺was 2.1 times that of low copy plasmids,and the background leakage level was only 1/10 of that of low copy plasmids.Under the same induction condition,the reporter protein SFGFP expression level was higher in the high copy plasmid sensor,so its dynamic output level was up to 466 times,nearly26 times higher than that of the low copy plasmid.Therefore,selecting plasmids with high copy number as expression vectors of gene circuits is a simple strategy to improve sensor performance.（2）Study on the influence of the location of the report module expression on the sensor performance:Compared with the intracellular expression,the orientation of the report module expression on the outer membrane is conducive to the direct measurement of the signal molecules of the report module.In this study,the reporter gene green fluorescent protein SFGFP was successfully expressed on the outer membrane of the cell surface by fusing with the anchored protein tag LOS-tag.However,due to the low expression level of fusion protein,the detection limit,dynamic response amplitude,and sensitivity of the sensor were not improved.Therefore,in addition to the directed expression of the reporter module on the membrane,improving the expression level of reporter genes on the membrane is the key to improve the performance of the sensor.（3）Study on the influence of performance optimization of report module on sensor performance:In this paper,a green fluorescent protein mutant（EGFP T203C S205C）with Hg²⁺-induced fluorescence enhancement effect was screened by structural simulation.The mutant had a good linear relationship in the range of 3-10μM,a detection limit of 0.5μM,and a fluorescence enhancement effect of up to 8 times of F/F₀ with good specificity.This binding site was introduced into the gene circuit as a reporter gene in order to construct a sensor with"double signal enhancement"mode.The results show that this whole-cell biosensor is reduced only at the level of background leakage.Therefore,in addition to improving the sensitivity of the reporter module to signal molecules,the expression of the reporter protein and the fluorescence intensity of the substrate also have a crucial impact on the performance of the sensor,which is an important research direction for later performance optimization.（4）Study on the influence of sensor module performance optimization on sensor performance:In this chapter,MerR mutants with stronger Hg²⁺binding ability（namely MerR V109E and MerR K99N）were used as transcription factors to construct two kinds of sensors.Compared with the wild-type MerR protein,the sensitivity and detection limits of the MerR V109E and MerR K99N sensors are greatly improved.The detection limit of the MerR V109E sensor is 0.4 n M,and the linear detection range is4～15 n M.It has good specificity and anti-interference in the presence of higher concentration of competitive ions.In addition,the Hg²⁺response was still good in a wide p H range（p H 4.0-10.0）.The results show that the performance of the sensor can be improved effectively by optimizing the performance of the sensor module.