Research On Construction Of A Heavy Metal Pb(?) Cell Biosensor And Its Optimal Design

Lead is one of the earliest metals applied by human beings.While lead brings convenience to production and life of mankind,its highly toxic bring serious damage to ecological environment and human health.The high levels of lead in environment often lead to the occurrence of mass lead poisoning.In this context,an efficient and rapid Pb?II?ion detection method has become the key to prevent laed poisoning.In this study,based on one metal binding protein and its specific promoter,we designed a variety of lead ion biosensor logic circuit.Through the determination of its performance,some high sensitivity cell biosensors of lead ion could be obtained.The main results of this paper are as follows:?a?According to the tests of the lowest and highest detection limits of the lead ion cell probes,all the 9 biosensors had a fluorescent response with the concentration of Pb?II?from 10^-2?M to 200?M.The results showed that all the lead ion sensor could be used to test the concentration of lead in drinking water in the range of the national standard.And moreover,the lowest detection limit of S231 and S232 could reach 10^-3?M.And the highest detection limit of S211,S231,D41,D42 and D43 can reach 300?M.?b?Based on the data analysis of lead ion cell biosensor,the artificial chassis with the highest fluorescence response range of Pb?II?concentration was S231.And the double-plasmid detection system had a better fluorescence response with the lead ion concentration from 50?M to 300?M.?c?By measuring the sensitivity and specificity of the biosensor,we found the influence of functions to the cells of the chassis by the detect component and the report component.And also,we obtained one efficient composite genetic circuit with the regulatory module P_pbr bi-directional controlling the functional modules including PbrR and fluorescent protein.We also obtained the amplification mechanism of fluorescent response by Lux R controlled.At the same time,we built and optimized the double-plasmid detection system of lead ion named D42 and D43.The optimized synthetic biology strategy of Lead ion cell biosensor could help to build a highly efficient artificial lead ion cell detection system.In this study,we designed a variety of genetic circuits by the core protein PbrR.And a series of performance tests on the sensitivity of lead ion detecition were did.By solving the key scientific problem of how to improve sensitivity and specificity of the sensor,we got an effective cell biosensor of heavy metal lead.And at the same time,our study provided a new idea of on-line identification and real-time detection of other heavy metal ions.