Gene replacement with linear DNA in electroporated wild-type Escherichia coli

Gene replacement using linear double-stranded DMA fragments in wild-type Es cherichia coli transformation is generally inefficient due to exonucleolyti c degradation of incoming DNA, Recombination-proficient strains, in which t he exonucleolytic activity of RecBCD is inactivated, have been used as tran sformation recipients to overcome this difficulty. Here we report that gene replacements using linear double-stranded donor DNA can be achieved in wil d-type E.coli if electrocompetent cells are used. Using a plasmid target, w e obtained 10(2)-10(3) gene replacement event/mu g linear DNA, Using an ind ependent chromosomal target, similar to 60 gene replacement events/mu g lin ear DNA were obtained, The presence of Chi sites on the linear DNA, which a re known to block DNA degradation and stimulate recombination in E.coli, ha d no effect on gene replacement efficiency in either case. RecBCD-mediated exonucleolytic activity was found to be diminished in electroporated cells, Electrotransformation thus provides a simple way to perform gene replaceme nts in many E.coli strains.