Topic Introduction

Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus

  1. Angelika Gründling3,4
  1. 1Microbiology, School of Biological and Chemical Sciences, University of Galway, Galway H91 TK33, Ireland
  2. 2Center for Pandemic Vaccines and Therapeutics (ZEPAI), Paul-Ehrlich-Institute, 63225 Langen, Germany
  3. 3Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, United Kingdom
  1. ↵4 Correspondence: a.grundling{at}imperial.ac.uk

Abstract

Here we describe an allelic-exchange procedure for the construction of an unmarked gene deletion in the bacterium Staphylococcus aureus. As a practical example, we outline the construction of a tagO gene deletion in S. aureus using the allelic-exchange plasmid pIMAY*. We first present the general principles of the allelic-exchange method, along with information on counterselectable markers. Furthermore, we summarize relevant cloning procedures, such as the splicing by overhang extension (SOE) polymerase chain reaction (PCR) and Gibson assembly methods, and we conclude by giving some general consideration to performing genetic modifications in S. aureus.

Footnotes

  • From the Experiments in Bacterial Genetics collection, edited by Lionello Bossi, Andrew Camilli, and Angelika Gründling.

Articles citing this article

  • Introduction of a Recombineering Oligonucleotide and a CRISPR-Cas9 Gene-Targeting Plasmid into Staphylococcus aureus for Generating a Gene-Deletion Strain Cold Spring Harb Protoc; 2024;
  • Using CRISPR-Cas9-Based Methods for Genome Editing in Staphylococcus aureus Cold Spring Harb Protoc; 2024;
  • Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli Cold Spring Harb Protoc; 2023;
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A more recent Protocol discussing this method is available

  • Topic Introduction Identifying cis-Acting DNA Elements within a Control Region
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    • and Stephen T. Smale
    Cold Spring Harb Protoc; 2012; doi:10.1101/pdb.top068171

This Article

  1. Published in Advance April 28, 2023, doi: 10.1101/pdb.top107945 Cold Spring Harb Protoc © 2023 Cold Spring Harbor Laboratory Press
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ORCID

  1. Profile for Gründling, A.http://orcid.org/0000-0002-6235-8687

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