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Schematic of cloning and generation of bivalent recombinant BCG strain. pBRL8 vector was digested with NotI/ClaI to remove lysA cassette (STEP 1) and the s1pt gene was PCR amplified and cloned under PL5 promoter thus generating pBRL-S1 vector (STEP 2). This vector was used to transform wild-type BCG (STEP 3) thus generating rBCG-S1i. In the STEP 4, rBCG-S1PT was made electrocompetent and used in a 2nd transformation step with pBRL-S1 to generate the bivalent strain.
