*2.4. In Vitro Stability of the BCG.HTIint Strain*

Five subcultures (∼35 bacterial generations) from the MS of BCG.HTI2auxo.int harboring the p2auxo.HTIint plasmid DNA (two selected clones) containing the lysine complementing gene were cultured in 7H9 Middlebrook broth with and without L-lysine selection. Subcultures were performed every 7 days by transferring 100 μL of the stationary phase culture to 5 mL of fresh medium. PCR analysis of the HTI DNA coding sequence were performed using insert specific primers designed at both HTI 3' and 5' sequences. PCR product size from the p2auxo.HTI.int plasmid and subcultures were compared.
