| Three whole-cell adsorbents for remediating the pollution of mercury and cadmium in the environment were constructed in this study.At first, the recombinant fusion gene ptet-inp-mt3a-egfp, ptet-inp-merR-egfp and pmerR-inp-merR-egfp were constructed respectively, containing the constitutive expression promoter ptet, Hg2+inducible expression promoter pmerR, ice nucleoprotein INP, Cd2+metallothionein Mt3a and Hg2+metal-binding protein MerR, and enhanced green fluorescent protein EGFP as a fluorescent marker. The recombinant fusion gene was inserted into the genome of Pseudomonas putida X4to get three corresponding strains.X4-1:X4/(ptet-INP-Mt3a-EGFP)X4-2:X4/(ptet-INP-MerR-EGFP)X4-3:X4/(pmerR-INP-MerR-EGFP)The expression of the fusion gene was proved under a fluorescence microscope. Through the Western Blot, we confirmed that the fusion protein has been displayed onto the cell surface.With the growth of strains X4-1, X4-2, the expression level of fusion protein was also increasing steadily. But X4-3exhibited a higher fluorescence intensity values in2h induced by2μM Hg2+, and the detectable concentration range of Hg2+ was10nM-100μM at28℃. The specificity of promoter is very strong, there was an extremely weak fluorescence when X4-3was induced by Cu2+, Cd2+and Zn2+.The maximum cadmium ion adsorption capacity of X4-1was11.2mg/g,3.1times of the original X4. The maximum mercury ion adsorption capacity of X4-2, X4-3was8.29mg/g and7.75mg/g,2.3and2.2times of the original X4, respectively. The recombinant strains have improved the adsorption capacity of heavy metal significantly.The successful application of these engineering strains would play a good role to monitor and repair heavy metal pollution in the soil and water. |
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