Cloneo in vivo de genes de Escherichia coli K12 mediante recombinacion homologa / Cloning of gene of Escherichia coli K12 by means of homologue recombination

RESUMO

Plasmid pT153, a stable recombinant between pBR322 plasmid and M13 bacteriophage, and Tn10 transposon were employed for in vivo cloning of a chromosomal segment of Escherichia coli including the nar locus. The strategy consisted in creating an homology between pT153 and the E. coli chromosome, incorporating a Tn10 transposon close to the nar locus of a polA12 temperature-sensitive strain. The selection of clones carrying the plasmid into the chromosome was realized at 42 degrees C, with ampiciline, taken advantage that the replicon of pT153 requires the ADN Polymerase I for its functioning. The plasmid integrated in the polA12 strain has the opportunity of excising and carrying part of bacterial genome and autonomically replicating after a thermal change from 42 degrees C to 30 degrees C. The stable recombinant plasmids could be efficiently transduced with the M13 bacteriophage to a E. coli strain (delta narGHJI), obtaining Nit+ Apr transductant clones. The versatility of this method of E. coli gene cloning is the facility to create the homologue region anywhere in the bacterial genome and the efficient transduction of pT153 plasmid by M13 bacteriophage