TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Description or sequence | Antibiotic resistance | Reference or source |
|---|---|---|---|
| L. lactis subsp. cremoris strains | |||
| MG1363 | Plasmid-free derivative of NCDO712 | 6 | |
| MP401 | MG1363 containing pMAP50 | Ermr | 23 |
| AJ189 | MG1363 containing pAJ149 (PL-lacLM) | Ermr | 13 |
| AJ218 | MG1363 containing pAJ155 (PR-lacLM) | Ermr | 13 |
| Escherichia coli strains | |||
| DH5α | supE44 hsdR17 deoR recA1 endA1 lacZΔM15 | Stratagene | |
| M15[pREP4] | Expression host for pQE vectors, contains pREP4 | Kanr | Qiagen |
| Plasmids | |||
| pCI372 | Shuttle vector | Camr | 8 |
| pAJ149 | pAK80::P3 (3142-3458)a PL-lacLM fusion, containing OR and OL | Ermr | 13 |
| pAJ155 | pAK80::P3 (3142-3458)a PR-lacLM fusion, containing OR and OL | Ermr | 13 |
| pAJ80 | pCI372::PRORcI (2622-3332) | Camr | 16 |
| pMAP50 | pAK80::cI+OR+OL+mor+OD+, PL-lacLM | Ermr | 23 |
| pMAP131 | pMAP50;15 base pairs inserted at codon 120 in cI | Ermr | This study |
| pMAP132 | pMAP50;15 base pairs inserted at codon 98 in cI | Ermr | This study |
| pMAP133 | pMAP50;15 base pairs inserted at codon 135 in cI | Ermr | This study |
| pMAP140 | pMAP50;15 base pairs inserted at codon 78 in cI | Ermr | This study |
| pMAP141 | pMAP50;15 base pairs inserted at codon 89 in cI | Ermr | This study |
| pMAP150 | pCI372::PRcI; 15 base pairs inserted at codon 78 | Camr | This study |
| pMAP151 | pCI372::PRcI; 15 base pairs inserted at codon 89 | Camr | This study |
| Oligonucleotides | |||
| MLN6.rev | 5′-GGGGGGGTTTAAACATTTTAATTGAATTTTTGCAGTATC-3′ | ||
| morBamHI.rev | 5′-GGGGGATCCATGAGTTATGATTATTCGTCAC-3′ | ||
| MLN6.for | 5′-GGGGGGGTTTAAACATAAAAGAACAAGATGAGCAAAATAAAGTTA-3′ | ||
| pAK80erm | 5′-CTTTCAACTGCCTGGCAC-3′ | ||
| MLN8.rev | 5′-GGGGGGGTTTAAACAAACTGTAATAGTTTCTTCTATATT-3′ | ||
| MLN8.for | 5′-GGGGGGGTTTAAACACAGTTATGAAAAAATTGGAAGAGCCAAGGC-3′ | ||
| MLN27.rev | 5′-GGGGGGGTTTAAACATCAGACGGTAATCTTCTATTTGTT-3′ | ||
| MLN27.for | 5′-GGGGGGGTTTAAACATCTGAGTGATGAATATCTTGAAGAACAAAT-3 | ||
| MLN2.rev | 5′-GGGGGGGTTTAAACATACATCAAAGCCCATTAACCACGC-3′ | ||
| MLN2.for | 5′-GGGGGGGTTTAAACACAGTTCCTATGGTAGAATCTTCTAAAATA-3 | ||
| MLN3.rev | 5′-GGGGGGGTTTAAACAATCATTTTCTATTTTAGAAGATTC-3′ | ||
| MLN3.for | 5′-GGGGGGGTTRAAACACAGTTTCTGAAAATATAGAAGAAACTATT-3 | ||
| CIBamHI | 5′-GGGGGGGATCCGCTCTCTAAGTTTACTCATAAATCG-3 | ||
| erm2BamH | 5′-CGGGATCCCGCACGTTTCATGAACTTT-3′ |
a
Numbers indicate nucleotide positions in the TP901-1 genome.