SyrB1, SyrB2, SyrB2 mutants, and Sfp were all expressed using the same method. Plasmids containing the proteins were transformed into BL21(DE3) cells (Thermo Scientific) for protein overexpression. An overnight culture (~50 mL 2XYT broth, 0.05 mg/mL kanamycin) was grown at 37°C for ~16 hours at 220 RPM. Ten mL of the overnight culture was used to inoculate 1 L cultures (2XYT, 0.05 mg/L kanamycin) in 2.8L baffled flasks. A single drop of Antifoam 204 (Sigma) was added to each culture to aid in oxygen diffusion. Cultures were grown at 37°C until an OD600 of 0.6-0.8 was reached. Cultures were then placed in an ice bath for 15-20 minutes to cool. Isopropyl β-D-1-thiogalactopyranoside (IPTG, GoldBio) was add ed to a final concentration of 0.2 mM to induce protein overexpression. After 18 hrs of shaking at 18°C, cell pellets were harvested by centrifugation, flash frozen in liquid nitrogen, and stored at -20°C.