A SUICIDE VECTOR FOR CONSTRUCTING A LYSA INSERTION MUTATION IN ESCHERICHIA COLI

Abstract

The objective of this study was to construct an insertion mutation in the lysA gene of Escherichia coli and investigate the effect of the insertion mutation on the growth rate response. The lysA gene encodes the last enzyme in the lysine biosynthetic pathway in most bacteria. A suicide plasmid pXL1 carrying a segment of the lysA sequence was transformed into E. coli SM10 that is lysogenic for λ pir. The plasmid was extracted from the transformant and confirmed to contain the segment of lysA by restriction enzyme digestion. After this confirmation, pXL1 was transferred by conjugation to an E. coli strain that cannot support replication of the plasmid. Maintenance of the selectable drug marker on the plasmid requires that the plasmid integrate into the chromosome at the lysA locus. The constructed mutant could grow in the absence of lysine supplementation although its growth rate was significantly (P < 0.05) depressed when compared to the parent strain.