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Gene replacement techniques for Escherichia coli genome modification

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Abstract

The subject of this review covers modern experimental procedures for chromosomal gene replacement in Escherichia coli and related bacteria, which enable the specific substitution of targeted genome sequences with copies of those carrying defined mutations. Two principal methods for gene replacement were established. The first “in–out” method is based on integration of plasmid into bacterial chromosome and subsequent resolving of the cointegrate. The “linear fragment” method (recombineering) is based on homologous recombination mediated by short homology arms at the ends of linear DNA molecule. Many new protocols and improvements in targeted gene replacement were introduced during the last 10 years. These methods are well suited for high-throughput functional gene studies and for many biotechnological applications.

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Abbreviations

λ Red:

Genes gam, bet, and exo from bacteriophage λ mediating linear DNA recombination

FLP:

Site-specific flip recombinase of the 2-μ plasmid of Saccharomyces cerevisiae

FRT:

FLP recombinase recognition site

Cre:

Site-specific recombinase from bacteriophage P1

loxP:

Cre recombinase recognition site

BAC:

Vector(s), bacterial artificial chromosome(s)

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Acknowledgments

This publication is the result of project implementation: development of the center of excellence for utilization of information on bio-macromolecules in disease prevention and in improvement of quality of life (ITMS 26240120027) supported by the Research & Development Operational Program funded by the ERDF. The work was also supported by the grant of Slovak grant agency VEGA 1/0344/10.

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  1. Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynská dolina, 842 15, Bratislava, Slovak Republic

    Mahesh Madyagol, Hend Al-Alami, Zdeno Levarski, Hana Drahovská, Ján Turňa & Stanislav Stuchlík

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  1. Mahesh Madyagol
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  2. Hend Al-Alami
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  3. Zdeno Levarski
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  6. Stanislav Stuchlík
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Correspondence to Stanislav Stuchlík.

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Madyagol, M., Al-Alami, H., Levarski, Z. et al. Gene replacement techniques for Escherichia coli genome modification. Folia Microbiol 56, 253–263 (2011). https://doi.org/10.1007/s12223-011-0035-z

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