Table 2.
Primers used in this study.
| Primer1 | Sequence (5′–3′)2,3 | Plasmid construction and function | Restriction site | Source or reference |
|---|---|---|---|---|
| RNA silencing in O. oeni | ||||
| ASctsR3 | CGTCCCGGGATGGCAGAAGCTAATATTTCAGAT | pSIPSYNASCtsR: ctsR coding sequence for antisense cloning in pSIPSYN | SmaI | This study |
| ASctsR6 | GGGCCATGGCAGATGCTGTGTATTGATTATCCA | NcoI | This study | |
| Heterologous expression and reporter system in B. subtilis | ||||
| ctsR2 | CGGACTAAGCTTTTATCCATGAATGTTTGTACTCT | pXTCtsR25: O. oeni ctsR coding sequence into pXT for heterologous expression in B. subtilis pETCtsR: O. oeni CtsR production | HindIII | This study |
| ctsR5 | CCGGGAGGATCCAAAGGAGGGGGTTGAATG | BamHI | This study | |
| Olcg16 | ATCGGCGGATCCTATCAAATACCTCCTATTAACTAA | pDLhsp18: hsp18 promoter region for transcriptional fusion with bgaB in B. subtilis | BamHI | (Grandvalet et al., 2005) |
| Olcg20 | GCCCGAATTCTAAATTAATCGAAGCCTTTTGAC | EcoRI | ||
| Olcg302 | GGGCCATGGCAAATACCTCCTGATTCATTAATGCAGGGGTAC | pSIPSYN: Synthetic promoter PSY N amplification | NcoI | (Darsonval et al., 2016) |
| Olcg303 | CCCAAGCTTGCGCAACTGTTGGGAAGGG | HindIII | ||
| Over-expression in E. coli BL21 | ||||
| Olcg1 | ATGCATGCCATGGCAGAAGCTAATATTTCAG | pETctsR : O. oeni ctsR coding sequence into pET28a(+) for overexpression in E. coli BL21 | NcoI | This study |
| Olcg2 | GGGCTCGAGTCCATGAATGTTTGTACTCTCA | XhoI | ||
| hsp18N | GGGCCATGGCAAATGAATTAATGGATAGA | pEThsp18 : O. oeni hsp18 coding sequence into pET28a(+) for overexpression in E. coli BL21 | NcoI | This study |
| hsp18S | GGGGAGCTCTTATTGGATTTCAATATGATGAGT | SacI | ||
| Bacterial Two-Hybrid system in E. coli | ||||
| CtsR21 | GGAGGATCCCGCAGAAGCTAATATTTCAGATTT | ctsR coding sequence,T18-ctsR and T25-ctsR fusions | BamHI | This study |
| CtsR20 | GGTGGTCTCGAATTCTTATCCATGAATGTTTGTACTCTCAA | EcoRI/BsaI | ||
| CtsR25 | AAGAAGCTTATTGATAGGAGGATCAAATTATGGCAGAAGCTAATA | ctsR coding sequence, ctsR-T18 and ctsR-T25fusions | HindIII | This study |
| CtsR22 | GGAGGATCCTCTCCATGAATGTTTGTACTCTCAA | BamHI | ||
| ClpL1-21 | GGAGGATCCCGCTAATAATGATTATTTTAATAACAG | clpL1 coding sequence,T18-ctsR and T25-ctsR fusions | BamHI | This study |
| ClpL1-20 | CTCGAATTCTCACTTTCCAGTGATCTTAATTG | EcoRI | ||
| ClpL1-23 | GGTGGTCTCAAGCTTATTGATAGGAGGATTCAATCATG | clpL1 coding sequence, clpL1-T18 and clpL1-T25fusions | HindIII/BsaI | This study |
| ClpL1-22 | GGAGGATCCTCCTTTCCAGTGATCTTAATTGTTC | BamHI | ||
| ClpL2-21 | GGTGGTCTCGGATCCCGCTGATTATAATGACGATCCCT | clpL2 coding sequence,T18- clpL2 and T25-clpL2 fusions | BamHI/BsaI | This study |
| ClpL2-20 | GGTGGTCTCGAATTCTTAATTATCTTTTTGATTCGTGGCGG | EcoRI/BsaI | ||
| ClpL2-27 | GGTGGTCTCAAGCTTAGGAGGAGGAAAAAATGGCTGATTATAATGACGATCCCT | clpL2 coding sequence, clpL2-T18 and clpL2-T25 fusions | HindIII/BsaI | This study |
| ClpL2-22 | GGTGGTCTCGGATCCTCATTATCTTTTTGATTCGTGGCGG | BamHI/BsaI | ||
| ClpC21 | GGTGGTCTCCTGCAGGATAATCAATACACAGCATCTG | clpC coding sequence, T25-clpC fusions | PstI | This study |
| ClpC20 | GGTGGTCTCTCTAGATTATTTTGTACTTTTTTCAATTTGGTG | XbaI | ||
| ClpC21C | GGTGGTCTCCTGCAGCGATAATCAATACACAGCATCTG | clpC coding sequence (with clpC20), T18-clpC fusions | PstI | This study |
| ClpC21KT | GGTGGTCTCCTGCAGCCGATAATCAATACACAGCATCTG | clpC coding sequence (with clpC20), T25-clpC fusions | PstI | This study |
| ClpC27 | CCCAAGCTTAGGAGGAAACATTCATGGATAATCAATACACAGCATCTG | clpC coding sequence, clpC-T18 and clpC-T25 fusions | HindIII | This study |
| ClpC24 | GGTGGTCTCCTGCAGTCTTTTGTACTTTTTTCAATTTGGTG | PstI | ||
1Forward primers are numbered with odd numbers and Reverse primers with even numbers; 2Restriction sites are underlined; and 3ATG codons are in italic and STOP codon appears in bold.