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

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