A rapid and simple method for inactivating chromosomal genes in Yersinia

Authors

  • Anne Derbise,

    Corresponding author
    1. Unité de Bactériologie Moléculaire et Médicale, Laboratoire des Yersinia, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
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    • 1

      These authors contributed equally to this work.

  • Biliana Lesic,

    1. Unité de Bactériologie Moléculaire et Médicale, Laboratoire des Yersinia, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
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    • 1

      These authors contributed equally to this work.

  • Denis Dacheux,

    1. Unité de Bactériologie Moléculaire et Médicale, Laboratoire des Yersinia, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
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  • Jean Marc Ghigo,

    1. Groupe de génétique des biofilms, Institut Pasteur, Paris, France
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  • Elisabeth Carniel

    1. Unité de Bactériologie Moléculaire et Médicale, Laboratoire des Yersinia, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
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*Corresponding author. Tel.: 33 (1) 45688335; Fax: 33 (1) 40613001, E-mail address: aderbise@pasteur.fr

Abstract

A polymerase chain reaction (PCR)-based procedure without any cloning step was developed for a rapid mutagenesis/deletion of chromosomal target genes in Yersinia. For this purpose, a PCR fragment carrying an antibiotic resistance gene flanked by regions homologous to the target locus is electroporated into a recipient strain expressing the highly proficient homologous recombination system encoded by plasmid pKOBEG-sacB. Two PCR procedures were tested to generate an amplification product formed of an antibiotic resistance gene flanked by short (55 bp) or long (500 bp) homology extensions. Using this method, three chromosomal loci were successfully disrupted in Yersinia pseudotuberculosis. The use of this technique allows rapid and efficient large-scale mutagenesis of Yersinia target chromosomal genes.

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