TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristics, construction, or nucleotide sequence (5′ → 3′)a | Reference or source |
|---|---|---|
| Strains | ||
| B. lactis DSM 10140 | Wild type | 22 |
| E. coli XL1-Blue | recA1 lac endA1 gyrA96 thi hsdR17 supE44 relA1 (F′ proAB lacIq lacZΔM15 Tn10 Tetr) | 3 |
| E. coli BL21(DE3)/pLysS | F−ompT hsdSB(rB− mB−) gal dcm (DE3)/pLysS (Cmr) | Novagen |
| Plasmids | ||
| pUC18 | Ampr LacZ′; cloning vector; 2.7 kb | 35 |
| pCL1920 | Spcr Strr; cloning vector; 4.6 kb | 19 |
| pGEM-T Easy | Ampr LacZ′; annealed T in both 3′ ends after linearization with EcoRV; 3.0 kb | Promega |
| pET-28a(+) | Kanr LacI; expression vector; 5.4 kb | Novagen |
| pFPK1 | Ampr; 2.62-kb BamHI-BamHI fragment from strain DSM 10140 in pUC18; 5.3 kb | This study |
| pFPK2 | Ampr; 1.6-kb PCR-derived fragment from strain DSM 10140 in pGEM-T Easy; 4.6 kb | This study |
| pFPK3 | Spcr Strr; 0.85-kb BamHI-SmaI fragment from pFPK2 insertion in pCL1920; 5.4 kb | This study |
| pFPK4 | Spcr Strr; 2.62-kb BamHI-insertion from pFPK1 in the BamHI site of pFPK3; 8.1 kb | This study |
| pFPK5 | Kanr; 2.5-kb PCR-derived fragment from strain DSM 10140 in pET-28a(+); 7.9 kb | This study |
| Oligonucleotidesb | ||
| pk5 | 5′-GGIACICCITGGCARAAR-3′ (974–991) | This study |
| pk6 | 5′-ATATATATARTAYTTRTCCATICCIATIAT-3′ (1019–1039 reverse)c | This study |
| pk7 | 5′-CATGGCAGAAGCTGGATCGTCCGGT-3′ (981–1005) | This study |
| pk9 | 5′-GCGAGATCCCGTGGCGT-3′ (2526–2542) | This study |
| pk15 | 5′-ATATATGAATTCATGACTAATCCTGTTATTGGTACC-3′ (956–979)c | This study |
| pk16 | 5′-AATTACAAGCTTTCACTCGTTGTCGCCGGCGG-3′ (3414–3433 reverse)c | This study |
a
Position in the nucleotide sequence according to the numbering in the GenBank database.
b
Oligonucleotides were synthesized by Microsynth AG, Balgach, Switzerland.
c
The underlined sequences correspond to an introduced tail for cloning purposes.