Escherichia coli genome targeting I. Cre-Zox-mediated in vitro generation of ori^- plasmids and their in vivo chromosomal integration and retrieval

We have constructed a plasmid system designed for the insertion of cloned DNA (e.g., genes, gene fusions, regulatory elements, etc.) into the Escherichia coli genome. Its principal feature is the presence of two tandem lox sites on the plasmids, which upon Cre-mediated in vitro recombination resolve the plasmids into ori^- and ori⁺ DNA circles. The non-replicating ori^- circles contain the λ, attP site, several unique restriction sites for cloning, a NotI site and Km^R, a kanamycin-resistance-encoding gene. The ori⁺ circles carry the origin of DNA replication (ori) together with several cleavage sites not present in the ori^- circles, including the rare site for the very efficient I-SceI enzyme, that are used to inactivate the ori⁺ circles and any unresolved plasmid DNA. We have used this system to insert cloned DNA into the host genome at (i) the attB site, by Int-mediated integration and (ii) at any predetermined sequence, as mediated by the Rec system(s) of the host. The genomes of the resulting transformants were analyzed by Nod digestion of the chromosomal DNA, embedded in agarose microbeads, followed by pulsed-field gel electrophoresis. A system for the retrieval of DNA fragments inserted at the attB site was also developed.