pEThCRM

Protocol tips

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	The resulting pEThCRM plasmid (S2 Fig) was transformed into ClearColi BL21(DE3) (Lucigen) cells using the heat shock method, and transformants were selected on an LB-kanamycin agar plate. Recombinant cells harboring pEThCRM were grown in LB with shaking at 200 rpm, 37°C with 30 μg/ml kanamycin until the OD600 reached 0.6. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the culture medium at 0.5 mM to induce protein expression, and cultures were incubated at 37°C for an additional 2 h.

Protocol tips

The resulting pEThCRM plasmid (S2 Fig) was transformed into ClearColi BL21(DE3) (Lucigen) cells using the heat shock method, and transformants were selected on an LB-kanamycin agar plate. Recombinant cells harboring pEThCRM were grown in LB with shaking at 200 rpm, 37°C with 30 μg/ml kanamycin until the OD600 reached 0.6. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the culture medium at 0.5 mM to induce protein expression, and cultures were incubated at 37°C for an additional 2 h.

Publication protocol

The synthetic crm197 gene (1,611bp) was optimized for E. coli codon usage (GenScript, Piscataway, NJ) (S1 Fig). The synthetic crm197 gene was cloned into pET28a(+) (Novagen) using NdeI and HindIII restriction enzyme sites. The gene was designed to include at the 5’ end, oligonucleotide sequences (66 bp) encoding a 6x polyhistidine tag and an enterokinase cleavage site. Cloning procedures were performed according to standard techniques. The resulting pEThCRM plasmid (S2 Fig) was transformed into ClearColi BL21(DE3) (Lucigen) cells using the heat shock method, and transformants were selected on an LB-kanamycin agar plate. Recombinant cells harboring pEThCRM were grown in LB with shaking at 200 rpm, 37°C with 30 μg/ml kanamycin until the OD600 reached 0.6. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to the culture medium at 0.5 mM to induce protein expression, and cultures were incubated at 37°C for an additional 2 h.

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