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High ferrous oxidation activity of Thiobacillus ferrooxidans genetically engineered bacteria build

Author: LiuZuo
Tutor: LinJianQiang;LinJianQun
School: Shandong University
Course: Microbiology
Keywords: Biohydrometallurgy Thiobacillus ferrooxidans Of Fe2 oxidation of activity rus gene cyc1 gene cyc2 gene Quantitative PCR Conjugative transfer
CLC: Q939.97
Type: PhD thesis
Year: 2010
Downloads: 201
Quote: 0
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Abstract


Acidophilic Thiobacillus ferrooxidans (Acidithiobacillus ferrooxidans referred to as Thiobacillus ferrooxidans) plays an important role in bacteria is a microbiological metallurgy, is currently the most studied bioleaching bacteria one. Thiobacillus ferrooxidans is a very the representative extremely acidophilic strict autotrophic bacteria can acidic conditions Fe2 is oxidized to Fe3, or elemental sulfur or reducing oxidation of sulfides to sulfuric acid, and rely on these inorganics The oxidation process for energy. The bacteria in bacteria metallurgy, coal desulfurization, waste water treatment, heavy metals in the sludge leaching and other aspects play an important role. Thiobacillus ferrooxidans can under acidic conditions Fe2 oxide Fe3, Fe3 as a strong oxidant, under acidic conditions, the metal compound oxidation of the sulfide minerals dissolve, this is a crucial step in the reaction in the bioleaching process Therefore, the ability of Thiobacillus ferrooxidans oxidation of Fe2 closely related to its ability to leaching. The genetic engineering of the Thiobacillus ferrooxidans Fe2 oxidative capacity transformation should be based on its the oxidation the Fe2 pathway of research. Acidophilic Thiobacillus ferrooxidans oxidation of Fe2 mainly through Fe2 electronic delivery system. Fe2 electron transport system, have raised a variety of models. Currently, with the most experimental evidence to support the most widely accepted electronic pathway model Fe2 → the protein by Cyc2 of cytochrome - of ceruloplasmin Rusticyanin → cytochrome Cycl → aa3 cytochrome c oxidase → 02. The gene encoding the electron transport chain of the component proteins are located within the same operon, the operon is named rus operon the IncQ family to the broad host range plasmid pJRD215 based construct containing Ptac strong promoter and an electronic transmission body reorganization of protein-coding genes the plasmid pTRUS, pTCYC1 well pTCYC2, and successfully imported Thiobacillus ferrooxidans ATCC19859 and thus constructed the the Thiobacillus ferrooxidans genetic engineering the bacteria A.ferrooxidans (pTRUS), A.ferrooxidans (pTCYC1) as well as A.ferrooxidans (pTCYC2). The Western-blotting validation, the recombinant plasmid carrying electron transfer body encoding gene in Thiobacillus ferrooxidans expression. In order to test the the Thiobacillus ferrooxidans of Fe2 oxidation activity the way, we detect Thiobacillus ferrooxidans genetically engineered bacteria build Fe2 oxidase activity is genetically engineered to improve. The experimental results show that, either resting cells or cell-free extracts of the test results, the the Thiobacillus ferrooxidans gene the engineered bacteria A.ferrooxidans (pTRUS) of A.ferrooxidans (pTCYC1) Fe2 oxidation activity have improved to some extent. In resting cells, the the Thiobacillus ferrooxidans genetic engineering the bacteria A.ferrooxidans (pTRUS) Ferrous A.ferrooxidans (pTCYC1) and A.ferrooxidans (pTCYC2) oxidase activity compared to the original strain ATCC19859, were increased by 19.85 %, 13.30% and 7.68%, respectively. In cell-free extracts, relative to the original strain of Thiobacillus ferrooxidans ATCC19859, ferroxidase activity genetically engineered bacteria A.ferrooxidans (pTRUS), and A.ferrooxidans (pTCYC1) increased by 41.41% and 38.57%, respectively; genetic engineering little difference bacteria A.ferrooxidans (pTCYC2) cell extract ferrous oxidase activity as measured with the original strain. To further explore the reasons for the improvement of genetically engineered bacteria Fe2 oxidation activity, we apply quantitative PCR technique composed of genetic engineering in the rus operon gene expression levels. The results showed that in genetically engineered bacteria 4.ferrooxidans (pTRUS) and A.ferrooxidans (pTCYC1), except for improved expression levels of the plasmid carried by the objective gene, the RUS to manipulation of the child on the other the level of gene expression can also have a certain improved. Changes in the level of the sub-gene expression through the manipulation of genetically engineered bacteria in rus initial the Thiobacillus ferrooxidans Fe2 oxidation electronic delivery system the rate-limiting step speculated provide theoretical guidance for the the Thiobacillus ferrooxidans further genetically modified. Thiobacillus ferrooxidans the ferrous medium the Fe2 consumption and growth of bacteria test results show that, in the the Fe2 medium of Thiobacillus ferrooxidans A.ferrooxidans (pTRUS) and A.ferrooxidans (pTCYC1) consumption Fe2 speed relative to the control strain has been significantly improved. Nevertheless, genetically engineered bacteria and control bacteria growth curve, but with no significant difference.

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