Allelic-Exchange Procedure in Staphylococcus aureus .

This protocol continues a series of methods for the construction of an in-frame gene deletion in strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-Δ) can be constructed and isolated from , then introduced into electrocompetent cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (* gene) and confers chloramphenicol resistance to As a specific example, we present the construction of strain RN4220*Δ from strain RN4220 carrying the pIMAY*-Δ plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.