Table 1.
Primers, plasmids and bacterial species used in this work
| Sequence (5′–3′) | Primer name | Restriction site |
|---|---|---|
| Primers used to amplify and clone ORFs under hsp60 | ||
| GGAGAATTCATGGAGTCCGAACCGCTG | BCG_0114-5F | EcoRI |
| CCCAAGCTTTTACGTGGCCGAGCC | BCG_0114-3R | HindIII |
| GTGCAGCTGTCATGGTAAAGGTCTTCTTGGTCG | devR-F-Pvu | PvuII |
| ACTAAGCTTCCTGTTGTCATGGTCCATCACCG | devR-RHd3 | HindIII |
| ACTGAATTCATGTCTGCTACGATACC | BCG_0642c-5F | EcoRI |
| GCCAAGCTTTCACCACCG | BCG_0642c-3R | HindIII |
| CAAGAATTCATGCGACACATGAGT | BCG_3766c-5F | EcoRI |
| GGTAAGCTTTTACGGAGCGGG | BCG_3766c-3R | HindIII |
| GCTGAATTCATGACCACCTCC | EthR-5F | EcoRI |
| GAGAAGCTTTTAGCGGTTCTCG | EthR-3R | HindIII |
| Sequence (5′–3′) | Primer name | |
|---|---|---|
| Primers used for real time qPCR | ||
| AGTCCGAACCGCTGTACAAG | BCG0114-5RT | |
| CAGCAGCTCCAAAATCCTGATC | BCG0114-3RT | |
| ATGGCAACGGCATTGAACT | dosR-F | |
| AGAATCGCATCTAGCATGGC | dosR-R | |
| GTAATCGCAGATCAGCAACG | rrs-F | |
| TTCGGGTGTTACCGACTTTC | rrs-R | |
| ATCTGAAACCCCAACACCAC | BCG_3766c-5RT | |
| ATAGCCGGTGAAGAAGATGACC | BCG_3766c-3RT | |
| TGGTCAATCAAGCCGACATG | ethR-5RT | |
| TCTCGAAGAACACGTTGATCCC | ethR-3RT | |
| ATCGCGCGAAAAACCATCTG | sigA-F | |
| TGGTGTAGTCGAACTTCTCCAC | sigA-R | |
| AGGAAATCGAGGTGCTCAAGG | BCG_0642c-5RT | |
| TCGCCCAGATTGGTGGTATC | BCG_0642c-3RT | |
| Plasmid | Description | |
|---|---|---|
| pMV361 | Shuttle vector, kanamycin resistance marker, incorporates as single copy into Mycobacterium attB site, can drive gene transcription from the strong hsp60 promoter | |
| pMV361-dosR | pMV361 with dosR inserted into PvuII/HindIII sites | |
| pMV361-BCG0114 | pMV361 with BCG0114 inserted into EcoRI/HindIII sites | |
| pMV361-BCG0642c | pMV361 with BCG0642c inserted into EcoRI/HindIII sites | |
| pMV361-BCG3766c | pMV361 with BCG3766c inserted into EcoRI/HindIII sites | |
| pMV361-ethR | pMV361 with ethR inserted into EcoRI/HindIII sites | |
| Strain | Genotype | |
|---|---|---|
| M. bovis BCG Pasteur | Mycobacterium bovis Karlson and Lessel (ATCC® 35734™) (hereafter referred as to BCG wild type -WT-) | |
| BCG WT::pMV361 | BCG Pasteur with an insertion at attB of pMV361 (empty vector) | |
| BCG::dosR | BCG Pasteur with an insertion at attB of pMV361-dosR | |
| BCG::BCG0114 | BCG Pasteur with an insertion at attB of pMV361-BCG0114 | |
| BCG::BCG0642c | BCG Pasteur with an insertion at attB of pMV361-BCG0642c | |
| BCG::BCG3766c | BCG Pasteur with an insertion at attB of pMV361-BCG3766c | |
| BCG::ethR | BCG Pasteur with an insertion at attB of pMV361-ethR | |
| E. coli DH5α | F− φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK−, mK+) phoA supE44 λ− thi-1 gyrA96 relA1 | |
Underlined sequences indicate sites for endonuclease restriction