Minimal Medium for Protein Production

Minimal medium protein production for growth and in vitro experiments were performed using M9 minimal medium (including casamino acids). The final composition of the M9 base medium was 1× M9 salts, 0.4% glucose, 0.5% Casamino acids, 1 mM MgSO₄, 100 μM CaCl₂, 36 μM FeCl_3, and ampicillin (100 μg/ml). The final composition of the sulfur-depleted M9 (M9-S) medium was 1× M9 salts, 0.4% glucose, 0.1% Casamino acids, 1 mM MgCl₂, 100 μM CaCl₂, 36 μM FeCl₃, and ampicillin (100 μg/ml). The conditions for protein production were the same as described above. For the M9 + 5 mM MgSO₄ and M9 + 5 mM l-cysteine experiments, cultures were moved to 18°C for 2 hours after induction followed by the addition of MgSO₄ or l-cysteine. Induced cultures were grown for 20 hours at 18°C.