The leuB or leuC gene was knocked out by introducing a premature stop codon using the CTBEevolve plasmid. The strains were grown in Middlebrook 7H9 broth supplemented with ADC and leucine to the stationary phase. A fraction of the cells was plated onto a Middlebrook 7H10 agar plate supplemented with ADC without leucine and another fraction was plated onto a Middlebrook 7H10 agar supplemented with ADC and leucine. The plates were sealed by parafilm and incubated for 4 days at 37°C.