Of Thermophilic Ethanol Bacteria Of Genetic Transformation Technology Research

Thermoanaerobacter ethanolicus JW200 is a Gram-positive, anaerobic thermophile that can grow at high temperature and in acidic conditions,it produce considerable amounts of ethanol from a wide range of polymeric and soluble carbohydrates. These advantages make this species an attractive candidate for use in bioconversion processes. Due to the low background interference and reliable results, in vitro transcription becomes a powerful tool for transcript analysis.Electrotransformation of T. ethanolicus JW200 was achieved using the integrated plasmid pTEI2.And we explored the method of electrotransformation and Ultrasound-mediated transformation of JW200, by the method of the transformation, we tried to find the change of ethanol production of the JW200.In order to find the replicon from the Gram-positive bacteriums which can stay stativly in JW200,the application of Tma DNA ligase was explored in PCR. Tma DNA ligase was employed in PCR cycles, Tma DNA ligase promoted the lingation of long DNA fragments. Taq DNA polymerase mediated error-prone PCR using whole plasmid as template has been developed for creating random mutagenesis libraries. In this megaprimer error-prone PCR reaction system, random mutations were introduced randomly during error-prone PCR reaction using Taq DNA polymerase. Circular plasmid was synthesized when the nick was ligated by Tma DNA ligase. The ligated circular plasmids were transformed into competent cells of Thermoanaerobacter ethanolicus JW200 and the transformants were spread by medium containing resistance.As an unkown protein, electrophoretic mobility shift assay showed that Teth11 could bind with promoter of adhE specifically. Further, we added it in vitro transcription system, and the result indicated that Teth11 inhibited the transcription of adhE. we predicted the structure of Teth11 on line. Two conserved domain was found in the animo acids sequence, the first one is between the eight and the eleven amino acid from the N extremity, the second one is between the Sixty-eight and Seventy-two amino acid from the N extremity.The helix-turn-helix motif was formed in its tertiary structure.The second conserved domain is part of the helix-turn-helix motif so we mutated the amino acid of the domain, and we verified the function of the protein by electrophoretic mobility shift assay of the mutated protein.