Quantification of mRNA levels of vanRS and vanHAX was done as described previously. Primer sequences for qPCR were also described previously.3 (link)
For quantification of gDNA levels of vanRS and vanHAX, DNA was extracted from VVESwe-S, VVESwe-R and BM4147 grown to mid-log-phase in BHI broth without and with 8 mg/L vancomycin, using the GenElute Bacterial Genomic DNA Kit (Sigma Aldrich). qPCR was performed using probes with 5′FAM and a 3′BHQ-1 quencher (Eurogentec), qPCR Master Mix Plus Low ROX (Eurogentec) and run on a 7300 Fast Real-Time PCR System (Applied Biosystems). Cycle threshold (Ct) values were normalized to the housekeeping gene gdh and ΔCt was calculated as ΔCtvanRS = CtvanRS−Ctgdh and ΔCtvanHAX = CtvanHAX−Ctgdh. The plasmid copy number was calculated as 2ΔCt(vanHAX) since only one copy of gdh is found in all strains, and vanHAX only localizes to the plasmid of interest (based on Lee et al.24 (link)).
Statistical data analysis of qPCR data was performed in GraphPad Prism 7 using an unpaired two-tailed t-test.
For quantification of gDNA levels of vanRS and vanHAX, DNA was extracted from VVESwe-S, VVESwe-R and BM4147 grown to mid-log-phase in BHI broth without and with 8 mg/L vancomycin, using the GenElute Bacterial Genomic DNA Kit (Sigma Aldrich). qPCR was performed using probes with 5′FAM and a 3′BHQ-1 quencher (Eurogentec), qPCR Master Mix Plus Low ROX (Eurogentec) and run on a 7300 Fast Real-Time PCR System (Applied Biosystems). Cycle threshold (Ct) values were normalized to the housekeeping gene gdh and ΔCt was calculated as ΔCtvanRS = CtvanRS−Ctgdh and ΔCtvanHAX = CtvanHAX−Ctgdh. The plasmid copy number was calculated as 2ΔCt(vanHAX) since only one copy of gdh is found in all strains, and vanHAX only localizes to the plasmid of interest (based on Lee et al.24 (link)).
Statistical data analysis of qPCR data was performed in GraphPad Prism 7 using an unpaired two-tailed t-test.
