Cultivation of Mycobacterium bovis BCG

Mycobacterium bovis BCG strain was cultured in a 2 liter bioreactor under growth conditions (Table EV1). Cultures were grown as batch for 7 days. Continuous cultures were grown under chemostat conditions at a growth rate of 0.03 h⁻¹ maintained by the media flow rate (Beste et al, 2011 (link)). Media was pumped into the chemostat using a peristaltic pump. Cultures were grown for 3–4 volume changes in the unlabeled media to assure a metabolic steady‐state before introducing isotopically labeled media. [¹³C₃] glycerol (12.5%) and [¹⁵N₁] NH₄Cl (20%) were the carbon and nitrogen isotopically labeled substrates in the media. Isotopic stationary state was assessed by measuring % label in the proteinogenic amino acids of cultures drawn at different times during label feed (Appendix Fig S2C).