Preparation of ElectrocompetentStaphylococcus aureusCells and Plasmid Transformation

Abstract

This protocol is part of a series of methodologies for the construction of an in-frame gene deletion inStaphylococcus aureusstrain RN4220. Having previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated fromEscherichia coli, we now present details of the next steps in this method—the preparation of electrocompetentS. aureuscells and introduction of thetagOmutant plasmid DNA into theS. aureuscells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.