*3.3. Phenotypic Characterization of BCG.HTI2auxo.int*

To preserve plasmid stability, both in vivo and in vitro, as well as to prevent potential genetic rearrangements, several factors should be considered when constructing mycobacterium-based vaccine candidates. We previously demonstrated that the use of weak promoters (*Mycobacteria spp*. α-antigen promoter) and use of the BCG lysine auxotrophy-complementation system prevent the disruption of gene expression due to genetic rearrangements [33,37]. The lysine auxotrophic BCG strain was used in combination with lysine gene complementation as an antibiotic-free plasmid selection and maintenance system. To phenotypically assess the stability and demonstrate the lack of antibiotic resistance of this system, the BCG.HTI2auxo.int strain was cultured on non-lysine-supplemented agar with and without kanamycin. In line with previous findings, the untransformed lysine auxotrophic BCG strain failed to grow without the presence of lysine and grew upon supplementation with lysine (Figure 3A,B). The BCG.HTI2auxo.int strain, on the other hand, grew on non-lysine supplementation (Figure 3C), and did not grow on agar plates containing kanamycin (Figure 3D).

**Figure 3.** *Cont*.

**Figure 3.** Phenotypic characterization of the BCG.HTI2auxo.int vaccine strain. Phenotype of lysine auxotrophy, lysine complementation, and kanamycin resistance. The BCG lysine auxotroph prior to transformation with p2auxo.HTIint was cultured on non-lysine supplemented 7H10 (**A**) or on lysine supplemented 7H10 (**B**). The BCG.HTI2auxo.int WVS was cultured on 7H10 without lysine or kanamycin supplementation or without lysine but with kanamycin supplementation (**C**,**D**, respectively).
