Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome

György Pósfai, Vitaliy Kolisnychenko, Zsuzsa Bereczki, Frederick R. Blattner

Research output: Contribution to journalArticle

181 Citations (Scopus)


A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the targeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I-SceI, is inserted into the genome by homologous recombination between the mutant and the wild-type (wt) alleles. Resolution of this cointegrate by intramolecular recombination of the allele pair results in either a mutant or a wt chromosome which can be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the chromosome at the I-SceI site. Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E. coli cells, does not require specific growth conditions, and is potentially applicable in other microorganisms. Use of the method was demonstrated by constructing a 17-bp and a 62-kb deletion in the MG1655 chromosome. Cleavage of the chromosome induces the SOS response but does not lead to an increased mutation rate.

Original languageEnglish
Pages (from-to)4409-4415
Number of pages7
JournalNucleic acids research
Issue number22
Publication statusPublished - Nov 15 1999

ASJC Scopus subject areas

  • Genetics

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