Bacteria from genus Prevotella are Gram negative, non-spore forming, obligate anaerobes that represent important part of gastrointestinal and oral microbiota of animal and humans. Members of the genus have been isolated from oral cavity of mammals, rumen of cattle, from large intestine, urogenital tract and clinical samples. They are the most abundant Gram negative bacteria in rumen and are contributing to the degradation of plant cell-wall polysaccharides and protein metabolism. The genetic background of the genus is poorly studied because there are not many developed genetic tools. As far as we know only one recipient strain Prevotella bryantii TC1-1 can be electrotransformed. In vitro shuttle vector pRH3 must be protected from restriction endonuclease with cell extract before electroporation. Electrotransformation of three strain P. bryantii and P. brevis was performed, although it wasn’t successful, mainly because of their strong nonspecific nuclease activity. We continued with finding optimal conditions for protection and electroporation in process of electrotransformation of strain P. bryantii TC1-1. With insertion of methylase gene PbrTI (locus_tag CIK91_RS00145) from P. bryantii into strain TC1-1 we tried to improve in vitro protection of DNA with cell extract. Protection of plasmid DNA wasn't successful. By isolation of the protein with Ni-NTA agarose affinity chromatography we tried to substitute preparation of cell extract, but the efficiency of protection of plasmid DNA wasn't successful, because proteolysis occurred.
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