TABLE 1.
Bacterial strains, plasmids, and oligonucleotide primers used in this study
| Strain, plasmid, or primer | Relevant characteristic(s), description, or sequencea | Source or reference |
|---|---|---|
| Strains | ||
| E. coli DH5α | supE44 ΔlacU169 φ80lacZΔM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | Novagen |
| E. coli BL21(DE3) | F−ompT hsdSB(rB− mB−) gal dcm (DE3) | Novagen |
| E. coli Origami B(DE3) | F−ompT hsdSB(rB− mB−) gal dcm lacY1 ahpC gor522::Tn10 (Tcr) trxB::Kan (DE3) | Novagen |
| L. lactis NZ9000 | MG1363 pepN::nisRK; most commonly used host for NICE system | O. P. Kuipers |
| Lb. plantarum K10 | Wild type | Our lab |
| Lb. brevis K31 | Wild type | Our lab |
| Lb. casei BL23 | Kind gift from S. Hazebrouck | 10 |
| S. thermophilus K11 | Wild type | Our lab |
| Lb. helveticus K17 | Wild type | Our lab |
| Lb. buchneri K43 | Wild type | Our lab |
| Lb. fermentum K37 | Wild type | Our lab |
| Lb. delbrueckii subsp. bulgaricus ATCC 11842 | Kind gift from M. van de Guchte | 27 |
| Plasmids | ||
| pBluescript-SK-lysin | pBluescript SK ligated to 1,257-bp PCR product containing lyb5 gene | 30 |
| pET-22b (+) | Apr; E. coli expression vector | Novagen |
| pWaldo-Control | pET-28b ligated to 725-bp GFPuv gene at NdeI and HindIII sites | 29 |
| pUC-bga | pUC ligated to 2,000-bp PCR product containing bga gene | 14 |
| pET-ly5C | Apr; pET-22b (+) carrying HindIII/XhoI-digested product expressing His6-tagged Ly5C | This study |
| pET-gfp | Apr; pET-22b (+) carrying NdeI/HindIII-digested product expressing His6-tagged GFP | This study |
| pET-gfp-ly5C | Apr; pET-22b (+) carrying NdeI/XhoI-digested product expressing His6-tagged GFP-Ly5C | This study |
| pET-gal | Apr; pET-22b (+) carrying NdeI/HindIII-digested product expressing His6-tagged β-Gal | This study |
| pET-gal-ly5C | Apr; pET-22b (+) carrying NdeI/XhoI-digested product expressing His6-tagged β-Gal-Ly5C | This study |
| Primers | ||
| LM1 | 5′-ACTAAGCTTTCTAGACACCAGGACACGCACAAC-3′ | This study |
| LM2 | 5′-GTGCTCGAGATAGTAGAGAGTTTGGCCG-3′ | This study |
| GFP-HindIII | 5′-GCGAAGCTTATGAGCAAAGGAGAAG-3′ | This study |
| GFP-XhoI | 5′-ACACTCGAGGTAGAGCTCATCCATG-3′ | This study |
| LM-NdeI | 5′-CGACATATGTCTAGACACCAGGACA-3′ | This study |
| LM-HindIII | 5′-GTGAAGCTTATAGTAGAGAGTTTGG-3′ | This study |
| Gal1 | 5′-CATCATATGATTAGCAGCAAATTACC-3′ | This study |
| Gal2 | 5′-AACAAGCTTCATCTCAAGCAGCTGAAC-3′ | This study |
a
The restriction sites in the primer sequences are underlined. Tcr, tetracycline resistance; Kan, kanamycin resistance; Apr, ampicillin resistance; NICE system, nisin-controlled gene expression system.