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Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system

Biological Procedures Online volume 8, pages 153–162 (2006)Cite this article

Abstract

In this work we describe protocols for the generation of gene deletions and gene replacements using a temperature sensitive plasmid in Escherichia coli O157:H7. This technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. A number of examples are used to illustrate the flexibility of the system which has been used to create novel gene replacements including fusions for protein localisation work and reporters for transcriptional analyses. In this paper we describe protocols which can be used with a high degree of success when applied to E. coli O157. The deletion and replacement of the LEE4 operon of E. coli O157 is detailed to show the advantages and limitations of the technology.

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Authors and Affiliations

  1. Centre for Infectious Diseases, University of Edinburgh, The Chancellors Building, 49 Little France Crescent, EH16 4SB, United Kingdom

    James R. Emmerson, David L. Gally & Andrew J. Roe

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  1. James R. Emmerson
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  2. David L. Gally
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  3. Andrew J. Roe
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Correspondence to Andrew J. Roe.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Emmerson, J.R., Gally, D.L. & Roe, A.J. Generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Biol. Proced. Online 8, 153–162 (2006). https://doi.org/10.1251/bpo123

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  • DOI: https://doi.org/10.1251/bpo123

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Biological Procedures Online

ISSN: 1480-9222

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