A plasmid-based method to quantitate homologous recombination frequencies in gram-negative bacteria

A method is described which enables quantitative evaluation of the ability of gram-negative bacterial cells to perform homologous recombination between DNA molecules. This method is particularly useful in cases where the stringency of rec mutations is to be determined. The procedure is based on a wide-host-range vector (pRK404) in which two unequally truncated and overlapping fragments of the neo gene were cloned. When introduced into gram-negative bacteria either by transformation or by conjugation, molecules of this plasmid, pBX404-7, undergo unequal crossing-over leading to the restoration of a functional neo gene. The stringency of putative rec mutations can thus be determined by measuring the frequency at which kanamycin-resistant colonies appear in bacterial strains harboring pBX404-7.