S. aureus

Preparation and transformation of S. aureus electrocompetent cells followed the method of Schenk and Laddaga⁷⁴, though used TSBY (Tryptone soya broth [Oxoid] containing 2.5% yeast extract) in place of B2 medium. Briefly, bacteria were grown with vigorous aeration in TSBY to an OD₆₀₀ of 0.6, harvested by centrifugation, and washed three times in an equal volume of sterile, deionized water. Subsequent wash steps used decreasing volumes of 10% glycerol; first 1/5 the original culture volume, then 1/10, finally resuspending in ~1/32 volume and storing the resultant electrocompetent cells at −80 °C. For electroporation, 60 µL of electrocompetent cells were mixed with ≧1 µg of plasmid DNA in a 1 mm electroporation cuvette at room temperature and pulsed at 2.3 kV, 100 Ω, 25 μFD. Immediately after electroporation, 390 µL room temperature TSBY was added to the cells and incubated with aeration at 37 °C for 1–2 h, before plating onto tryptone soya agar with appropriate antibiotic selection. Using this method, sequence-verified constructs established in E. coli were first transferred into the restriction deficient S. aureus RN4220 strain⁷⁵, before recovery and introduction into S. aureus SH1000 (refs. ^76,77).