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Background and Purpose: ferrooxidans (Thiobacillus ferrooxidans, Tf) was isolated in 1947, a new strain, which is an obligate autotrophic and extremely acidophilic sulfur bacteria, which is widely distributed in soil, water, sulfur Fountain and deposition springs, widely used in low-grade copper ore, uranium and many other precious metals leaching and recovery, which is currently the most studied biological metallurgy, most cost-effective bioleaching microorganisms. However, since the slow-growing bacteria, the cell was low, and the uranium, fluoride ions and some lack of resistance, so that in practice limit its scope of application, but also on the research work to bring a lot of inconvenience. In this study, using molecular biology techniques, gene cloning anti-fluoride flr-4, to import T.f1 in order to obtain a stable genetic resistance to fluorine genetically engineered bacteria. Method: 1. Using RT-PCR technology, from Caenorhabditis elegans gene cloned anti-fluoride (flr-4), construct pET30a-flr-4 prokaryotic expression vector, import it into E. coli BL21 (DE3), using SDS- PAGE and Western blot analysis Flr-4 protein expression by growth kinetics, detection of E. coli resistance to fluorine changes in capacity. (2) Building pJRD215-flr-4 vector into E. coli SM10 after its use of conjugal transfer technology, vector into T.f1, detection pJRD215-flr-4 genetic stability, relatively wild-type and recombinant T T.f1 . f1 fluoride concentration of different strains grown under kinetic curves. Results: 1. Cloned genes resistant fluorine flr-4, prokaryotic expression vector pET30a-flr-4, to be introduced into E. coli BL21 (DE3) after the detection of E. coli in a specific fusion protein. Wild-type E. coli BL21 (DE3) in the NaF was 9.6g / L of TB medium growth was inhibited, while Flr-4 fusion protein expressed in recombinant E. coli BL21 (DE3) affected their growth. (2) Construction of a vector pJRD215-flr-4, by engaging the shift into T.f1, wild type T.f1 the NaF concentration was 40mg / L or more is no longer grow, and in the NaF concentration was 20mg / L of the time to reach steady to eight days, restructuring T.f1 in NaF concentration 60mg / L in the case could grow, and in the NaF concentration 20mg / L in the case of the original eight days to reach steady shortened four days. The recombinant T.f1 in the absence of selective pressure conditions, the continuous passage 50 generations, plasmid retained more than 70%. Conclusions: 1. By RT-PCR techniques from Caenorhabditis elegans gene cloned anti-fluoride flr-4, prokaryotic expression vector pET30a-flr-4, and transformed into E. coli BL21 (DE3) in. (2) by SDS-PAGE and Western blot technique detected Flr-4 fusion protein in E. coli BL21 (DE3) expression, and confirmed the expression of anti-fluoride enhances the ability of E. coli resistance to fluorine. 3 constructed pJRD215-flr-4 expression vector, the vector is introduced via conjugation to ferrooxidans T.f1, access to the genetic stability of anti-fluoride genetically engineered bacteria, T.f1 genetically engineered bacteria resistant fluorine capability increased by 200% or more.
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