Genetic System for Reversible Integration of DNA Constructs and lacZ Gene Fusions into the Escherichia coli Chromosome

Author： Platt R. Drescher C. Park S-K. Phillips G.J.

ISSN： 0147-619X

Source： Plasmid, Vol.43, Iss.1, 2000-01, pp. : 12-23

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Abstract

A plasmid system for site-specific integration into and excision and recovery of gene constructs and lacZ gene fusions from the Escherichia coli chromosome was developed. Plasmid suicide vectors utilizing the origin of replication of R6K plasmids and containing the attP sequence of bacteriophage λ, multiple cloning site, and antibiotic resistance markers facilitate reversible integration into the E. coli chromosome by site-specific recombination. Additional vectors permit construction of lacZ gene fusions in three possible reading frames for recombination with the bacterial chromosome. These suicide vectors can be propagated in newly constructed E. coli strains that harbor different pir alleles. Two helper plasmids that encode the necessary gene products for integration (Int) and excision (Int and Xis) were also 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.