pMyCA‐mCherry

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	One milliliter precultures were employed to inoculate 10 mL working cultures in 7H9 medium. Protein expression was induced at early log‐phase (equivalent to OD600 near 1.0) using 34 mM acetamide and an additional incubation period of 24 h post‐induction.

Protocol tips
One milliliter precultures were employed to inoculate 10 mL working cultures in 7H9 medium. Protein expression was induced at early log‐phase (equivalent to OD600 near 1.0) using 34 mM acetamide and an additional incubation period of 24 h post‐induction.

Publication protocol

"One milliliter precultures were employed to inoculate 10 mL working cultures in 7H9 medium. Protein expression was induced at early log‐phase (equivalent to OD600 near 1.0) using 34 mM acetamide and an additional incubation period of 24 h post‐induction. The bacteria were harvested by centrifugation at 3500g and 4°C for 10 min and washed once with 1 mL ice‐cold lysis buffer (pH 7.4) containing 20 mM sodium phosphate, 300 mM NaCl, 1 M urea and 20 mM imidazole.

Mycobacterium smegmatis cell pellets were suspended in cold lysis buffer at a ratio of 5 mL buffer per gram wet weight of the bacterial pellet. The buffer additionally included a mix of AEBSF [4(2‐Aminoethyl)benzenesulfonyl fluorid hydrochlorid] 104 µM, leupeptin 10.5 µM and E‐64 1.4 µM (all from Carl Roth) to inhibit bacterial proteases. The bacterial suspensions were then lysed by sonication with a LabSonic 1515 sonicator (B. Braun, Melsungen, Germany) for 1 min at 50 W. Nonsoluble components were separated from the lysate by centrifugation at 20,000g and 4°C for 30 min (Eppendorf Centrifuge 5417R). Afterwards, the clarified supernatants were filtered through a 0.45 µm cellulose acetate membrane syringe filter (Carl Roth). The proteins were visualized by either 15% or 10% SDS‐PAGE gels, run at 120 V for 70 min and stained with colloidal coomassie stain.31 Protein samples for electrophoresis containing mCherry were not heat‐denatured to prohibit the temperature dependent hydrolysis of the main chain acylimine linkage.32"

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