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This protocol outlines the transformation and cultivation procedures for BL21 and TG1 strains harboring pSNAP, pSORTc, pFc, pHEN2, and pSORTp plasmids. Key steps include preparing competent cells, transforming with antibiotics, growing cultures in LB and 2xTY media with glucose, and optimizing growth conditions using arabinose and IPTG induction. The methodology involves centrifugation and storing harvested bacterial cells at -20°C for future experiments. Troubleshooting tips for lack of colony formation are also included.
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Suppl. Figure 2 Vector of expression Cytoplasmic : pSNAP,pSORTc, pFc Periplasmic : pHEN2, pSORTp Transformation in BL21 + Sulfhydryl oxydase LB +antibiotics (ABs) Transformation in TG1 2 xTY +1%glucose+ AB No colonies No colonies Colonies Colonies Sample 2-3 colonies in 3 mL LB, grow ON at 30°C, 210 rpm Check ABs Competent cells Check ABs Competent cells Makeglycerol Stocks Prepare 5 mL ON culture LB + AB Prepare 30 mL ON culture 2xTY+ 1%glu+ AB Growat 30°C Growat 37°C Add 2mL pre-culture in 500 mL LB+ ABs Add 25mL pre-culture in 500 mL 2xTY+ 0.1%glu+ AB Growuntil OD600nm=0.4 Growuntil OD600nm=0.6 Add arabinose 0.5% w/v, decrease the temperature to 20°C Add IPTG 0.5mM Growuntil OD600nm=0.6 Growat 20°C Add IPTG 0.05mM Grow 4 h at 28°C Harvestbacteria by centrifugation at 4000 rpm, 15 min, 4°C freeze and store at -20°C Grow ON Harvestbacteria by centrifugation at 4000 rpm, 15 min, 4°C ,freeze and store at -20°C
