Allelic-Exchange Procedure 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, Exhibition Road, London, SW7 2AZ, United Kingdom
- ↵4Correspondence: a.grundling{at}imperial.ac.uk
Abstract
This protocol continues a series of methods for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for S. aureus. We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, then introduced into electrocompetent S. aureus cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (pheS* gene) and confers chloramphenicol resistance to S. aureus. As a specific example, we present the construction of strain RN4220*ΔtagO from strain RN4220 carrying the pIMAY*-ΔtagO plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.
Footnotes
-
From the Experiments in Bacterial Genetics collection, edited by Lionello Bossi, Andrew Camilli, and Angelika Gründling.
