Construction of aStaphylococcus aureusGene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery inEscherichia coli

Abstract

We present a protocol for the generation of a gene-deletion allelic-exchange plasmid and its recovery inEscherichia colifor the purpose of constructing an in-frame gene deletion inStaphylococcus aureus. Here, we present detailed methodologies for (i) the primer design (using theS. aureus tagOgene as our specific example); (ii) PCR amplification of the required gene fragments; (iii) preparation of the cloning vector (using theS. aureusallelic-exchange vector pIMAY* as an example); (iv) the Gibson assembly cloning method; (v) introduction of the plasmid intoE. coli; (vi) confirmation of the plasmid insert inE. coliby colony PCR; and, finally, (vii) confirmation of the insert by sequencing. We also consider the long-term storage of theE. colistrains containing the desired plasmid.