Cloning and Expression of ardA Gene

The ardA sequence was amplified using the primers ardA-fwd and ardA-rev (Supplementary Table S3) and genomic DNA from S. aureus BMB9393 as template. The ardA gene was initially cloned into the p-GEM T easy vector (Promega) and subcloned into the expression vector pCN40 using BamHI and EcoRI restriction sites (Charpentier et al., 2004 (link)). The recombinant plasmid pCN40A (pCN40:P_blaZ-ardA) or empty pCN40 were transformed by electroporation into DC10B competent cells (Monk et al., 2012 (link)). The recombinant plasmids (pCN40A) and pCN40 were obtained (QIAfilter Plasmid Midi Kit; Qiagen), and transformed into RN4220 by electroporation (Monk et al., 2012 (link)), yielding the clones 42P40E (RN4220; empty pCN40) and 42P40A (RN4220; pCN40A). Transformants were confirmed by DNA sequencing and the expression of ardA in 42P40E was detected using real-time qRT-qPCR.