Genetic system for reversible integration of DNA constructs and lacZ gene fusions into the Escherichia coli chromosome

A plasmid system for cite-specific integration into and excision and recove ry of gene constructs and faa gene fusions from the Escherichia coli chromo some was developed. Plasmid suicide vectors utilizing the origin of replica tion of R6K plasmids and containing the attP sequence of bacteriophage lamb da, multiple cloning site, and antibiotic resistance markers facilitate rev ersible integration into the E. coil chromosome by site-specific recombinat ion. Additional vectors permit construction of lacZ gene fusions in three p ossible reading frames for recombination with the bacterial chromosome. The se suicide vectors can be propagated in newly constructed E. coli strains t hat harbor different pb alleles. Two helper plasmids that encode the necess ary gene products for integration (Int) and excision (Int and Xis) were als o constructed. This plasmid system was shown to be a reliable and efficient means to integrate and subsequently recover plasmids from the E. coli attB site. a 2000 Academic Press.