Study On The Whole-cell Biosensors Of Polychlorinated Biphenyls

Polychlorinated biphenyls（PCBs）are a class of chlorinated biphenyl compounds,which are internationally recognized as one of the persistent organic pollutants.They have the characteristics of bioaccumulation,high toxicity,high stability,environmental durability,and large range migration.However,the current detection methods for PCBs are difficult to operate and costly,so it is urgent to develop a convenient,fast,low-cost and efficient detection method.With its advantages of fast response,low cost,simple operation and high accuracy,whole-cell biosensor has become a new powerful tool for detecting environmental pollutants.In this paper,the polychlorinated biphenyl whole-cell microbial sensor was constructed by combining the upstream pathway of aerobic degradation of polychlorinated biphenyls based on the bph operon derived from Burkholderia xenovorans LB400 and the sensing pathway of the fusion of Xyl S/Pm system derived from Pseudomonas putida TOL plasmid p WW0 and green fluorescent protein e GFP.The main results are as follows:1、After verifying E.coli DH5αUnder the condition of tolerance to 2m M of 2,2-dichlorobiphenyl,the upstream pathway of aerobic degradation of PCBs was successfully constructed.After 120h induction of 0.5m M of 2,2-dichlorobiphenyl,the degradation rate was56%;2、In order to improve the degradation ability of PCBs,the S283M mutant of Bph A1was designed according to the literature report,which increased the degradation rate by 11%.Comparing the three promoters,p UTR/P_J23100/P_WT,which affect the degradation pathway,the promoter p UTR has the best effect.Deletion of genes Bph R and orf1 increased the degradation rate by 5%,and the final degradation efficiency was 70%;In addition,in E.coli BL21（DE3）successfully constructed the upstream pathway of aerobic degradation of PCBs with a degradation rate of 63%.After deleting the genes Bph R and orf1,the degradation rate increased by 6%.The S283M mutant on Bph A1 was also designed,which increased the degradation rate by 11%,and the final degradation rate was 80%;3、In order to improve the sensitivity of chlorobenzoic acid sensing pathway,the-35 and-10 regions of Ps and Pm promoters were changed into the common sequences TTGACA and TATAAT in prokaryotes,After the change of Ps promoter,the overall fluorescence decreased by 27%～61%.After changing the-35 region of Pm promoter alone,the overall fluorescence value was similar to that of wild type,but the five mutants of St Ep13 had better broad-spectrum;4、Combining the upstream pathway of aerobic degradation of PCBs in with the chlorobenzoic acid sensing pathway which in E.coli DH5α,the response ability of the recombinant strain CB1X5 to the concentration of 2.2’-dichlorobenzene and 4.4’- dichlorobenzene at 0.01/0.05/0.1 m M was tested,and it was found that CB1X5 could not respond to the polychlorinated biphenyls at this concentration.Subsequently,the degradation products of the upstream pathway of aerobic degradation of PCBs were detected by using the chlorobenzoic acid sensing pathway,and the results still showed that CB1X5 could not respond.The next work still needs to focus on improving the degradation rate of PCBs.The four key enzymes Bph A,Bph B,Bph C and Bph D in the upstream pathway of aerobic degradation can be rationally designed to make them have higher activity.At the same time,the reporter gene should be replaced,and the sensitivity of the sensor should be enhanced by cascade amplification of signals to provide a more efficient PCBs whole-cell biosensor.