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

Abstract

Here we describe an allelic-exchange procedure for the construction of an unmarked gene deletion in the bacteriumStaphylococcus aureus. As a practical example, we outline the construction of atagOgene deletion inS. aureususing 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 inS. aureus.