ESCHERICHIA-COLI GENOME TARGETING .1. CRE-LOX-MEDIATED IN-VITRO GENERATION OF ORI(-) PLASMIDS AND THEIR IN-VIVO CHROMOSOMAL INTEGRATION ANDRETRIEVAL

We have constructed a plasmid system designed for the insertion of clo ned DNA (e.g., genes, gene fusions, regulatory elements, etc.) into th e Escherichia coli genome. Its principal feature is the presence of tw o tandem lox sites on the plasmids which upon Cre-mediated in vitro re combination resolve the plasmids into ori(-) and ori(+) DNA circles. T he non-replicating ori(-) circles contain the lambda attP site, severa l unique restriction sites for cloning, a NotI site and Km(R), a kanam ycin-resistance-encoding gene. The ori(+) circles carry the origin of DNA replication (ori(-)) together with several cleavage sites not pres ent in the ori(-) circles, including the rare site for the very effici ent I-SceI enzyme, that are used to inactivate the oni(-) 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 integra tion and (ii) at any predetermined sequence, as mediated by the Rec sy stem(s) of the host. The genomes of the resulting transformants were a nalyzed by NotI 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 dev eloped.