TABLE 2.
Primers used in this study
| Use | Name | Sequence (5′→3′) | Restriction site | Target | Comment |
|---|---|---|---|---|---|
| Protein expression | Ex-breD-F | CAACATATGGACAATAAAATTCGTGAATATC | NdeI | breD | |
| Ex-breD-R | AAACTCGAGTTAGAAAGACTTGGGATTAAA | XhoI | breD | ||
| Ex-breG-F | CACCATATGAATATTTCTCAGAAACTTAAAC | NdeI | breG | ||
| Ex-breG-R | CTCGAGCTAAAGTTTATTATCATTTTTATTC | XhoI | breG | ||
| EMSA | EMSA-breA-F | Biotin-TCACGCTCAGCTGCTGTTTT | Region I | ||
| EMSA-breA-R | TATTTTGGTCGCGGGTACCT | Region I | |||
| EMSA-breB-F | Biotin-CAGTTTAGAAAGGTTGTTAAATCGG | Region II | |||
| EMSA-breB-R | GAATTTCTCCATTTCGATCGCTC | Region II | |||
| EMSA-breE-F | Biotin-AGCTTTCATAGTAAAACCTCCCTC | Region III | |||
| EMSA-breE-R | GTGCAATTGATAAACTATGAATCGACG | Region III | |||
| EMSA-breG-F | Biotin-ATACGTTTGTCCAAGCAGTTGTC | Region IV | |||
| DNase I footprinting assay and primer extension | FP-breBC-F | TCTGTCCTTTAAAGACCAAAAAATT | Upstream of breB | ||
| FP-breBC-R | GAATTTCTCCATTTCGATCGCTCTC | Upstream of breB | |||
| Biotin-FP-breBC-F | Biotin-TCTGTCCTTTAAAGACCAAAAAATT | Upstream of breB | |||
| Biotin-FP-breBC-R | Biotin-GAATTTCTCCATTTCGATCGCTCTC | Upstream of breB | |||
| FP-breED-F | GTAATCATTGCTACGCCAAGTAATT | Upstream of breE | |||
| FP-breED-R | TAAACTATGAATCGACGATTCCGTT | Upstream of breE | |||
| Biotin-FP-breED-F | Biotin-GTAATCATTGCTACGCCAAGTAATT | Upstream of breE | |||
| Biotin-FP-breED-R | Biotin-TAAACTATGAATCGACGATTCCGTT | Upstream of breE | |||
| Vector construction for promoter assay | PA01 | TATACTGCAGAAAACGAGGCAATTTGCCTCGTTTTCTTTTTCACTGCCCGCTTTCCAGTC | PstI | For constructing pre-pLES-PA | |
| PA02 | GCATGCCTGCAGGTCGACTC | PstI | For constructing pre-pLES-PA | ||
| PA03 | CAGTGAATTCGAGCTCGGTACCCGGGGATCCTCT | EcoRI | For constructing pLES-PA | ||
| PA04 | TATAGAATTCCATATGATAGACGGTTTTTC | EcoRI | For constructing pLES-PA | ||
| PA-β-gal-F1 | TATAGGTACCAGTTGTTGACAGAATGGACATACTATGATATATTGTTGCTATAGCGTCACACAGGAAACAGCTATG | KpnI | β-Gal gene | Containing PLb sequence | |
| PA-β-gal-R1 | TATAGGATCCAAAAGAAAACGAGGCAAATTGCCTCGTTTTTAATGGATTTCCTTACGCGA | BamHI | β-Gal gene | Containing terminator sequence | |
| PA-β-gal-F2 | TATAGGTACCTCACACAGGAAACAGCTATG | KpnI | β-Gal gene | ||
| PA-β-gal-R2 | TATAGGATCCGCCCGGTTATTATTATTTTTG | BamHI | β-Gal gene | ||
| PA-PBreBC-F | TATAGGTACCAGACCAAAAAATTATAAATATATGG | KpnI | PBreBC | For pLES-PA-D and E | |
| PA-PBreBC-R | TATAGGTACCTTATTTTTTCCAAATGACGAATCAT | KpnI | PBreBC | For pLES-PA-D and E | |
| PA-PBreED-F | GAGCGGTACCAACTTTTAGATTTTTCATAATTAAT | KpnI | PBreED | For pLES-PA-B, C, and F | |
| PA-PBreED-R | TATAGGTACCAAATTTGCAATATATACACGTCATT | KpnI | PBreED | For pLES-PA-B, C, and F | |
| PA-BreD-F1 | TATAGGATCCGAGGCGAATCCAATGGACAATAAAA | BamHI | breD | For pLES-PA-C | |
| PA-BreD-F2 | AAAAGGATCCAGTTGTTGACAGAATGGACA | BamHI | breD | For pLES-PA-F | |
| PA-BreD-R | GCGCCTGCAGTTAGAAAGACTTGGGATTAAATAGA | PstI | breD | For pLES-PA-C and F | |
| PA-BreG-F | TTTTGGATCCATTAGAGGCATTCATGAACCATGAT | BamHI | breG | For pLES-PA-E | |
| PA-BreG-R | TATACTGCAGAGAATATACTTTTAAAGCCAGAATA | PstI | breG | For pLES-PA-E | |
| PA-β-gal-deletion-F | AACAGCTATGGACAATAAAATTCGT | For pLES-PA-F | |||
| PA-β-gal-deletion-R | TTGTCCATAGCTGTTTCCTGTGTGAC | For pLES-PA-F | |||
| Brevicin 174A biosynthetic gene cluster construction | IF_breA–H_insert-F | GGTACCCGGGGATCCAGTTATGGATTTGCACCAGAACCTA | Gene cluster | ||
| IF_breA–H_insert-R | CTTGCATGCCTGCAGTTAAACTACCAGACGCCAATTGAGG | Gene cluster | |||
| IF_breA–H_vector-F | CTGCAGGCATGCAAGCTTGG | pLES003-b | |||
| IF_breA–H_vector-R | GGATCCCCGGGTACCGAGCT | pLES003-b | |||
| breD_mutation-F | GAGGCGAATCCAATGGACAATAAATAGCGTGAATATCGGAAG | For breD mutation | |||
| breD_mutation-R | CTTCCGATATTCACGCTATTTATTGTCCATTGGATTCGCCTC | For breD mutation | |||
| breG_mutation-F | GATATGAATATTTCTCAGTAGCTTAAACAATGTCGTAGTGCC | For breG mutation | |||
| breG_mutation-R | GGCACTACGACATTGTTTAAGCTACTGAGAAATATTCATATC | For breG mutation | |||
| qRT-PCR | qPCR-breB-F | GAAATATACCGGACCAAACTACCG | breB | ||
| qPCR-breB-R | ACCACCAACGATACCACCAAC | breB | |||
| qPCR-breC-F | GGGGAAATGCAGCAACAG | breC | |||
| qPCR-breC-R | ATTGTGAAACGCCCCAGATAG | breC | |||
| qPCR-breE-F | ACCGAGAAGCCATAAAACACCT | breE | |||
| qPCR-breE-R | CGGCAACAAACGATCCAA | breE | |||
| qPCR-rep-F | GGCAGTCTAACACCGACCA | rep | |||
| qPCR-rep-R | CTTTGCGTTCAGGTTTCCA | rep | |||
| qPCR-gyrB-F | TGAATACCGAAAAGGCCAAA | gyrB | |||
| qPCR-gyrB-R | ACCAACACCCGCACCAA | gyrB | |||
| qPCR-β-gal-F | CAGGTAGCAGAGCGGGTAAA | β-Gal gene | |||
| qPCR-β-gal-R | ATCCCAGCGGTCAAAACA | β-Gal gene |