A rapid method for disrupting genes in the Escherichia coli genome.

The entire genomic sequence of Escherichia coli has recently been completed. To gain insight into the function of the vast array of yet uncharacterized open reading frames (ORFs), a variety of new genetic tools will be required. Here we examined a genetic system, using an integration plasmid vector (named pINT007), for rapid construction of disruption mutants of any ORF in E. coli. It was found that the vector allows us to rapidly construct a disruption mutant for any gene on the chromosome as a cointegrate, furthermore, resolution of the resulting cointegrate can be surely accomplished by using a pair of the bla (ampicillin resistant) genes on the vector as a positive-selection marker.