In vivo excision and amplification of large segments of the Escherichia coli genome

György Pósfai, Michael Koob, Zdenka Hradečná, Noaman Hasan, Marcin Filutowicz, Waclaw Szybalski

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

In vivo excision and amplification of large segments of a genome offer an alternative to heterologous DNA cloning. By obtaining predetermined 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 conditional replication origin (ori), is targeted by homologous recombination. The strain carrying the insertions is stably maintained until, upon induction of specifically engineered genes, the host cell expresses the site-specific recombinase and an ori-specific replication protein. 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 isolated as a large plasmid. The feasibility of such an approach is demonstrated here for E.coli. Using the yeast FLP/FRT site-specific recombination system and the π/γ-ori replication initiation of plasmid R6K, we have devised a procedure that should allow the isolation of virtually any segment of the E.coli genome. This was shown by excising, amplifying and isolating the 51-kb lacZ-phoB and the 110-kb dapX-dsdC region of the E.coli MG1655 genome.

Original languageEnglish
Pages (from-to)2392-2398
Number of pages7
JournalNucleic acids research
Volume22
Issue number12
DOIs
Publication statusPublished - Jun 25 1994

ASJC Scopus subject areas

  • Genetics

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