Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus
- 1Microbiology, School of Biological and Chemical Sciences, University of Galway, Galway H91 TK33, Ireland
- 2Center for Pandemic Vaccines and Therapeutics (ZEPAI), Paul-Ehrlich-Institute, 63225 Langen, Germany
- 3Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, United Kingdom
- ↵4Correspondence: 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
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From the Experiments in Bacterial Genetics collection, edited by Lionello Bossi, Andrew Camilli, and Angelika Gründling.
