The growing bacterial resistance to antibiotics calls for the elaboration of new pathogens elimination strategies. Some of these methods are based on the conjugative transfer of recombinant plasmids able to eliminate pathogenic recipients by plasmid run-away replication or by killing activity of plasmid-encoded bacteriocins. Using live bacteria as donors of plasmid vectors carrying killing determinants requires meeting many safety restrictions in order to eliminate potential biohazard.
The technology used for the cloning of the open reading frame (ORF) I and II reflecting DNA fragments in regard to the ligations of the inserts with the pET-3a vector and the identification of the bacterial colonies, containing the recombinant plasmids, is presented. The main steps include: calculation of the molar ratios of the vector to insert DNA, the ligation reactions, transformation of the competent E. coli cells of the XL-1 strain, isolation of the plasmids, digestion of the plasmid preparations with the Nde I and Bam HI to indicate the presence of the inserts cloned into the plasmids, using the agarose gel electrophoresis (2% gel, 1 x TBE buffer) of the plasmids samples, with the released inserts, after the digestion, transformation of the competent BL21 (DE3) cells of E. coli with the isolated recombinant plasmids, identification of the bacterial cells, carrying the pET-3a plasmids with the cloned inserts and the storage of these cells.
JavaScript jest wyłączony w Twojej przeglądarce internetowej. Włącz go, a następnie odśwież stronę, aby móc w pełni z niej korzystać.