Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli

Cold Spring Harb Protoc. 2023 Aug 1;2023(8):107946. doi: 10.1101/pdb.prot107946.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • Escherichia coli* / genetics
  • Gene Deletion
  • Genetic Vectors / genetics
  • Plasmids / genetics
  • Staphylococcus aureus* / genetics