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Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation
Accetto, Tomaž ; Peterka, Matjaž ; Avguštin, Gorazd
ǂThe ǂrestriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ...ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barries hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strains which contained 20mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides-Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain faciliated the transfer with further increases efficiency and made possible the introduction of ligation reaction products directy to P. bryantii TC1-1 without passing them first through E. coli.
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