96 well assay block

For c4 and the c4 variants, the MIC_Amp assay was performed as previously described^{9 (link),10 (link)}. BL21 cells were transformed with pDIMC8 plasmids containing genes that encode the protein variants. For each overnight culture, 5 mL of tryptone broth (10 g L⁻¹ tryptone and 10 g L⁻¹ NaCl) was inoculated by picking a single colony, and incubated in a 37°C shaker for 16–18 hrs. The optical density at 600 nm (OD_600nm) was measured. For the MIC_Amp assay, approximately 1×10⁶ colony forming units (based on OD_600nm) from the overnight culture were added to 1 or 5 mL of tryptone medium containing chloramphenicol (Cm, 50 μg mL⁻¹), isopropyl-β-D-thiogalactopyranoside (IPTG, 300 μM), Amp (0 to 8192 μg mL⁻¹), and either the absence or presence of 5 mM maltose in each culture tube. For temperature dependent MIC_Amp assay, 5 mL cultures were incubated in 18°C and 37°C shaker incubators for 36 hrs and 18 hrs respectively, and the OD_600nm of each culture was measured. For pH dependent MIC_Amp assay, pH of the tryptone medium was adjusted with HCl and NaOH, and 1mL cultures in 96-well assay block (Corning, Tewksbury, MA) were incubated in 37°C shaker incubators for 18 hrs. No growth was observed at pH 5. The MIC_Amp was defined as the lowest concentration of ampicillin at which the OD_600nm was < 5 % of the OD_600nm in the absence of ampicillin.