Construction And Expression Of Copper Resistance Whole-cell Sensors

The specific promoter Cop A and Cue R gene were amplified from DNA genome of Escherichia coli by polymerase chain reaction (PCR) respectively. In addition, we constructed the plasmid (CGPNPCT) utilizing the green fluorescent protein (GFP) as a report gene and pGEM as the basic plasmid vector. Then it was was transformed into E. coli DH5a host cell and induced to express the protein by adding copper ions. Finally, the engineering strain tolerance to copper and its derivatives was determined.Through these experiments, a bacterium strain with resistance to heavy metal copper was successfully constructed hereafter the effects of copper ion concentration and inducing time on fluorescence intensity of the engineering strain were studied. In addition, the nature of engineering bacteria was also characterized. Based on the work above, it allowed us to study the feasibility of engineering strains for rapid detection of heavy metal copper. Now the research results about engineering strain are as follows:1. According to the experiment, DH5a containing CGPNPCT had lower expression of green fluorescent protein in the absence of copper inducement. So we can use it as the control reference. Moreover, there was better expression with higher fluorescence intensity and absorption peak at 480-520 nm after 4 h inducement by Cu2+.2. Engineering strain DH5a (CGPNPCT) in LB medium was supplemented with different concentrations of Cu2+ and then induced for 4 h. The result showed that the fluorescence expression gradually increased with Cu2+ concentration raised, then began to decrease after the peak value at 1.5mmol/L of Cu2+, finally the balance occurred when the concentration of Cu2+ was 3mmol/L. The fluorescence intensity had a positive relation to copper concentration with 0～4 mmol/L. 3. Engineering strain DH5a (CGPNPCT) in the medium with the same final concentration of Cu2+ as the inducement was sampled to determined the fluorescence intensity every 2 h during 12 h. The results showed a pronounced upward trend. In other words, with the same concentration of Cu2+, there was steady increase for the fluorescence intensity over time and the fluorescence value was proportional to the time.4. Six kinds of heavy metals (Cu2+,As5+,Cd2+,Cr3+,Hg2+,Pb2+) were used as inducement, to examine specificity of the engineering strains for Cu2+ by measuring the fluorescence intensity. The results indicated that engineering strain had obvious response to Cu2+ but no response to other heavy metals, suggesting that engineering strain is special to Cu2+.Experimental results showed that microbial whole-cell sensor technology can be as a sensitive and accurate method for detection of heavy metal copper using the engineering strain DH5a (pGEM-CGP-Nptll-Cue-Te) which containing green fluorescent protein gene (gfp) as report gene. And this method would have a strong application prospect.