IN-VIVO EXCISION AND AMPLIFICATION OF LARGE SEGMENTS OF THE ESCHERICHIA-COLI GENOME

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermine d fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. This approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50 - 100 kb apart. The integration of these sequences, together with a c onditional replication origin (ori), is targeted by homologous recombi nation. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expres ses the site-specific recombinase and an ori-specific replication prot ein. The recombinase then excises and circularizes the genomic segment flanked by the two insertions. This excised DNA, which contains ori, is amplified with the aid of the replication protein and can be isolat ed as a large plasmid. The feasibility of such an approach is demonstr ated here for E.coli. Using the yeast FLP/FRT site-specific recombinat ion system and the pi/gamma-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtua lly any segment of the E.coli genome. This was shown by excising, ampl ifying and isolating the 51-kb lacZ- phoB and the 110-kb dapX - dsdC r egion of the E.coli MG1655 genome.