Search

Bakterije rodu Prevotella so po Gramu negativni, nesporogeni in anaerobni mikroorganizmi, ki so del prebavne ter ustne mikrobiote živali in ljudi. Vrste so izolirane iz ustne votline sesalcev, iz vampa prežvekovalcev, debelega črevesa, urogenitalnega trakta in kliničnih vzorcev. Vampne prevotele predstavljajo največji delež po Gramu negativnih bakterij v populaciji in prispevajo k razgradnji polisaharidov v rastlinski celični steni in metabolizmu proteinov. Orodja za genetsko manipulacijo prevotel so maloštevilna, prav tako ni na voljo orodja za tarčno inaktivacijo genov. Poznamo samo en sprejemni sev Prevotella bryantii TC1-1, za katerega je na voljo plazmidni vektor, ki nam omogoča kontrolirano izražanje genov. Za vnos v ta sev prenosljivi vektor pRH3 najprej in vitro zaščitimo pred restrikcijskimi endonukleazami in ga vnesemo v sev z elektroporacijo. Na enak način smo poskušali elektrotransformirati še tri druge seve P. bryantii in P. brevis, vendar nam ni uspelo, saj imajo sevi izraženo močno nespecifično nukleazno aktivnost. Nadeljevali smo z ugotavljanjem optimalnih pogojev elektrotransformacije P. bryantii TC1-1 in ugotovili najbolj primerne pogoje zaščite in elektroporacije. Z vnosom gena za metilazo PbrTI (locus_tag CIK91_RS00145) iz P. bryantii v sev TC1-1 smo želeli izboljšati metodo zaščite DNK s celičnim ekstraktom, a zaščita ni bila boljša. Z izolacijo proteina z afinitetno Ni-NTA kromatografijo smo želeli nadomestiti pripravo celičnega ekstrakta. Učinkovitost zaščite DNK z izoliranim proteinom prav tako ni bila uspešna, saj je med njegovo izolacijo potekla proteoliza. 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.