Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome

Abstract

The isolation and amplification of large, predetermined segments of a genome from its host have been explored. The prototype of our approach was the excisional replication of some viruses such as the λ-lysogen. Similar machinery was used to excise and amplify large genomic segments of Escherichia coli in its host. Two loxP sequences for a site-specific recombinase Cre, together with a conditional replication origin (π-dependent γ-ori), were inserted into the genome by homologous recombination at predetermined sites, 50–100 kb apart. Cre and pir200 which encodes the site-specific recombinase Cre and an ori-specific replication protein π, respectively, were also introduced into the genome. The predetermined genomic segments flanked by the loxP sequences were excised and amplified upon induction of the cre and pir200 genes which were under the control of the tet promoter. This excised and amplified DNA could be easily purified as a large plasmid. This procedure can provide an alternative to conventional cloning methods by obtaining predetermined large genomic segments directly from the original organisms. In this study, using the Cre/loxP site-specific recombination and π/γ-ori replication system of plasmid R6K, a procedure was devised that could isolate a large segment of the E. coli genome and demonstrated the feasibility of the procedure by excising and amplifying the 50-kb trg-narZ and 100-kb trg-hipA regions of the E. coli W3110 genome.