Cloning and Expression of VirF Protein

Protein expression was performed as described previously.^{24 (link),29 (link),30 (link)} The open reading frame of virF was cloned into pET15b using primers virFpet15 FP/RP and pET15 FP/RP listed in Table 2, by Gibson assembly. Clones were confirmed by PCR using primers pET15bchk FP/RP and DNA sequencing. Plasmids were electroporated into E. coli BL21 DE3 for expression. Cultures were grown to OD₆₀₀ of 0.6 and induced with 5 mM IPTG (Isopropyl-β-D-1-thiogalactopyranoside) and grown for four hours aerobically at 37°C. Cells were centrifuged and aliquots were run on 12% SDS-PAGE to confirm induction of protein expression. Post confirmation, pellets were dissolved in Xtractor buffer followed by sonication to lyse the cells. Talon beads (TaKaRa) were used to extract His-tagged VirF by affinity purification following Takara’s protocol. Elution of VirF was confirmed by SDS-PAGE. Eluted proteins were dialyzed overnight in dialysis buffer (50 mM sodium phosphate, 300 mM NaCl, 10% glycerol). Size and purity of the protein were confirmed by running aliquots on 12% SDS-PAGE. Protein concentrations were calculated in NanoDrop (Thermo Scientific).