*2.2. Bacterial Cultures and Transformation*

Cells of the glycine auxotrophic strain of *E. coli,* M15Δ*Gly*, provided by Dr. Pau Ferrer (Universitat Autònoma de Barcelona, Spain), were cultured in LB supplemented with glycine (70 μg/mL). The *E. coli* M15Δ*Gly* cells were transformed with the p2auxo.HTIint plasmid by electroporation. For this, the *E. coli* cultures were grown to an optical density of 0.125 at 600 nm, as well as concentrated and transformed using a Bio-Rad gene pulser electroporator at 2.5 kV, 25 μF, and 200 Ω. The transformed cells were subsequently cultured on M9-D agar plates (minimal M9-derivative medium: Na2HPO4, 6.78 g/L; KH2PO4,3g/L; NaCl, 0.5 g/L; NH4Cl, 1 g/L, glucose, 10 g/L; MgSO4, 2 mmol/L; CaCl2, 0.1 mmol/L; thiamine, 0.1 g/L; FeCl3, 0.025 g/L; AlCl3·6H2O, 0.13 mg/L; ZnSO4·7H2O, 2.6 mg/L; CoCl2·6H2O, 0.47 mg/L; CuSO4·H2O, 4.6 mg/L; H3BO3, 0.03 mg/L; MnCl2·4H2O, 4.2 mg/L; NiCl2·6H2O, 0.02 mg/L; Na2MoO4·2H2O, 0.06 mg/L, with 1.5% bactoagar added) without glycine supplementation for selection or with glycine supplementation as a control. The QIAprep Spin Miniprep Kit was used according to the manufacturer's instructions (Qiagen, Hilden, Germany) to extract plasmid DNA from *E. coli*. The resulting plasmids were tested for identity and correct insertion by PCR and restriction enzyme profiling. The selected plasmid was transformed into BCGΔ*lys*.

The lysine auxotrophic BCG strain, BCGΔ*lys*, kindly provided by W.R. Jacobs Jr., B.R. Bloom, and T. Hsu (Albert Einstein College of Medicine, New York, NY, USA), was transformed with p2auxo.HTIint plasmid by electroporation. The mycobacteria were cultured in Middlebrook 7H9 broth medium or on Middlebrook agar 7H10 medium supplemented with albumin–dextrose–catalase (ADC; Difco Laboratories, Franklin Lakes, NJ, USA) containing 0.05% Tween 80. L-lysine monohydrochloride (Sigma) was dissolved in distilled water and used as a supplement at a final concentration of 40 μg/mL. For transformation, BCG was cultured to an optical density of 1.5 at 600 nm, washed with 10% glycerol, concentrated, and transformed using a Bio-Rad gene pulser electroporator at 2.5 kV, 25 μF, and 1000 Ω. Then, the transformants were cultured on ADC-supplemented Middlebrook agar 7H10 medium containing 0.05% Tween 80 without lysine supplementation. The resulting colonies were assessed for plasmid insertion, integrity, and HTI expression. From a selected colony, a Master Seed (MS) and a Working Vaccine Stock (WVS) were produced according to the seed lot system. For the BCG substrain identification assay, the commercial BCG Danish 1331 strain (Pfizer, New York, NY, USA) kindly provided by Dr. Neus Altet (Urology Department at Hospital Clínic de Barcelona, Barcelona, Spain), and the commercial BCG Connaught strain (ImmuCyst, Aventis, Paris, France) were used as standards.
