The Establishment And Optimization Of Cell-free Arsenic Biosensor Based On Arsenic-Responsive Protein ArsR

Arsenic and its compounds are widely distributed persistent toxic pollutants in nature.Arsenic exposure can cause many diseases threatening human health.Arsenic pollution cannot be permanently eliminated by current technologies,therefore monitoring and detecting arsenic contamination are extremely important.Traditional methods for arsenic contamination detection depend on large chemical analyzer instruments with time consuming and low throughput and require trained professionals to carry out specialized laboratories.Bacterial whole-cell biosensor is a new detection technology developed in recent years.It is convenient,simple and sensitive.However,due to the interference of bacterial cell wall and cell membrane barrier,as well as the complex metabolism of the cell,the stability of the detection is poor.To solve the limitations of whole cell biosensor,the core genetically encoded component of whole-cell biosensor,arsenic response operon/reporter gene,were used to construct arsenic biosensor with cell-free protein synthesis system in vitro.The main research results are as follows:1、We screened the ArsR mutant which was sensitive to arsenic and suitable for expression in vitro from the ArsR mutant library.The results showed that ArsR mutant was responsive to arsenic(50 μg/L)in vitro,and the fluorescent expression of GPF was increased by 3.4 times.2、We found that hat ep3 ArsR and GFP proteins are expressed in vitro,and ArsR regulates the expression of the reporter gene GFP.3、The effects of component extraction and reaction conditions of cee-free system with different strength on the performance of arsenic cell-free biosensor were studied.The results showed that the best detection condition was 37℃,pH7.2,30 minutes,then adding arsenic trioxide standard solution to induce for 2 hours,the expression level of GFP was significantly increased.The cell extracts from E.coli Rosetta strain prepared by ultrasonic treatment maintained high activity,which was conducive to the transcriptional translation function of cell-free protein synthesis system in vitro.4、The effects of reporter gene and RBS binding sequence with different strength on the performance of arsenic cell-free biosensor were studied.We found that the basic dynamics of genetic circuit in cell-free system is different from that in living cell,and the effective design of genetic coding circuit is the key factor in the combination of cee-free protein synthesis system and biosensor technology.5、The cell-free arsenic biosensor has a good linear response between 0 and 50 μg/L,and the detection limit is 3.65 μg/L,which is less than the arsenic concentration(10μg/L)allowed by China drinking water quality standard.In this study,we established a cell-free arsenic biosensor method for arsenic detection,which provides a new idea for rapid and accurate detection of arsenic,and promotes the application of biosensor of cell-free protein synthesis system in the detection of environmental toxic pollutants.