| Based on E.coli DH5a strain which owned heavy metal resistance gene, a recombinant plasmid was constructed using the firefly luciferase (luc) gene as the reporter. A heavy metal resistance engineered strain was constructed successfully in virtue of the transfer of the recombinant plasmid into E.coli DH5a. The study mainly focused on the corresponding relationship between fluorescence intensity presented by luc-tagged bacteria and concentration of Arsenic-ion, further, the characteristic of the engineered strain, aiming to test its capacity of detecting Arsenic content fast. Meanwhile efforts were taken on for improving the engineered gene, in order to reduce the background expression and increase the sensitiveness of the engineered bacteria.The plasmid pUC18-Luc-arsR with firefly luciferase gene as reporter was constructed on the basis of pUC18 vector, and will express firefly luciferase induced by Arsenic-ion. The relative particular promoter, responding to heavy-metal Arsenic-ion, and the regulative protein O/P-arsR were obtained using PCR of the genome DNA of E.coli DH5a. The expression plasmid was transferred to E.coli DH5a bacteria being guest by using chemical conversion competences method, and the engineered strain aiming to detect the content of Arsenic-ion was constructed successfully. The results of research were as followed:1.O/P-arsR expression was induced by the AsⅢand AsⅤ, which were added into cultivated medium of the DH5a (pUC18-Luc-arsR). Fluorescence intensity enhanced with the increase of AsⅢconcentration, in the presence of less than 10μM AsⅢ. While it weakened with the increase of AsⅢconcentration in the presence of more than 10μM AsⅢ. In addition, Fluorescence intensity enhanced with the increase of AsⅤconcentration, in the presence of less than 100μM AsⅤ. While it weakened with the increase of AsⅤconcentration in the presence of more than 100μM AsⅤ. 2. The engineered strain had no answer to other ten heavy mental ions such as Cd2+,Co2+, Cu2+ , Fe2+, Mn2+, Pb2+, Zn2+, Sn2+, Sb3+,Bi3+ except for Arsenic-ion. According to the results obtained from adding various concentr -ation of AsⅢ,AsⅤin the medium for the engineered strain, the engineered strain was a particular biosensor for detection of Arsenic-ion.3. The content of Arsenic on the surface and inside of the engineering strain cells was determined by combination of scanning spectrum and liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP -MS), with the inducing Arsenic-ion concentration of 1mM. The content of Arsenic on the surface of cells: the one induced by sodium arsenite for 4h was higher than induced for 1h, and the one induced by Sodium arsenate tribasic for 4h was higher than for 1h. The content of different forms of Arsenic inside the cells: the total content of Arsenic inside the cells induced by Sodium arsenate tribasic for 1h was about 38ppb, almost equal to the one for 4h. The content of AsⅤwas more than AsⅢwhen it had been induced for 1h, on the contrary, AsⅢcontent was greater than AsⅤwhen it had been induced for 4h. The content of Arsenic induced by sodium arsenite for 1h was higher than for 4h.4. The recombinant plasmid pUC18-Luc-2O/P-arsR was improved by the introduction of O/P next to O/P-arsR sequence in order to reduce the background expression. As result, the background expression was reduced remarkably and the sensitiveness for detecting was enhanced obviously. The results showed that a convenient and quick method would be established to detect the heavy mental Arsenic by using DH5a (pUC18 -Luc-arsR) with firefly luciferase gene as reporter or by using the improved DH5a (pUC18-Luc-2O/P-ars), which owned great practical significance.
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