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Figure 2 | BMC Molecular Biology

Figure 2

From: Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria

Figure 2

Bivalent recombinant BCG strain containing two pAV6950-derived vectors inserted into the tRNAalaU and tRNAalaV T-loops. BCG was transformed with the two vectors – pNIP46, targeting tRNAalaV, and pBU-lacZ, targeting tRNAalaU – to obtain a strain coexpressing the two foreign genes. A: Expression of the lacZ gene in rBCG::pNIP46::pBU-lacZ was detected as blue coloration on agar plates supplemented with the β-galactosidase substrate X-gal. B: Expression of gag p26 was detected by western blotting and wild-type BCG (lane 1), rBCG::pNIP46, (lane 2) and three different clones of rBCG::pNIP46::pBU-lacZ (lanes 3, 4, 5) were analysed. Lane 6 corresponds to purified recombinant Gagp26 produced in E. coli with an N-terminal His-tag. C: Multiplex PCR using the three sets of primers located upstream and downstream from the three different attB sites in BCG. Absence of the 200 bp, 400 bp or 600 bp amplification products indicates disruption of the tRNAalaU, tRNAalaV and tRNAalaT T-loops, respectively. The three fragments were present in the wild-type BCG chromosome (lane 1). After a single integration of pBU-lacZ into tRNAalaU, only the 600 and 400 bp fragments were present (lane 2), and in BCG::pNIP46, in which tRNAalaV had been disrupted, both the 600 and 200 bp fragments were amplified (lane 3). pBT disrupted the tRNAalaT locus (lane 4). In the double integrant, BCG::pNIP46::pBU-lacZ, only the tRNAalaT locus was intact and gave the 600 bp amplification fragment (lane 5). D Integration of each vector was confirmed by PCR using a mixture of the three primers upstream from tRNAalaU, tRNAalaV and tRNAalaT combined with LM2, annealing to the Ms6 integrase. No amplification was detected in wild type BCG chromosome (lane 1), by contrast, single products were detected in BCG::pBU-lacZ (lane 2), BCG::pNIP46 (lane 3) and BCG::pBT (lane 4). In BCG::pNIP46::pBU-lacZ two bands were obtained confirming the double integration (lane 5). E A diagram of the double integration of pNIP46 and pBU-lacZ is provided

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