Cloning and Expression of Staphylococcus aureus Spot-tagged Proteins

The ptpA, ptpB, and secA genes were amplified by PCR using S. aureus Newman chromosomal DNA as a template, and the Spot fragment was amplified by PCR using the pSpot2 vector as a template (Chromotek, Planegg, Germany) with the primers listed in Table S1. The ptpA, ptpB, and secA plasmids were constructed using NEB Gibson Assembly kit (New England Biolabs, Ipswich, MA, USA). The ptpA, ptpB and secA purified PCR products were fused to the Spot-tag at the C-terminus and Gibson cloned into the KpnI/EcoRI digested pRMC2 vector [31 (link)], thus generating pRMC2_PtpA-Spot, pRMC2_PtpB-Spot and pRMC2_SecA-Spot, respectively. The plasmids were propagated in E. coli IM08B [28 (link)] and electroporated into S. aureus strain SA564 [27 (link)].