Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments.

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/mol BamHI pSa fragment carrying determinants of resistance to four antibiotics in the unique BamHI site of pNH602. The resulting in vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 per E. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in the BamHI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymes BamHI and EcoRI and its physical and genetic map was constructed.