Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli
- 1Microbiology, School of Biological and Chemical Sciences, University of Galway, Galway, H91 TK33, Ireland
- 2Center for Pandemic Vaccines and Therapeutics (ZEPAI), Paul-Ehrlich-Institute, 63225 Langen, Germany
- 3Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, Exhibition Road, London, SW7 2AZ, United Kingdom
- ↵4Correspondence: a.grundling{at}imperial.ac.uk
Abstract
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.
Footnotes
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From the Experiments in Bacterial Genetics collection, edited by Lionello Bossi, Andrew Camilli, and Angelika Gründling.
