Table 1.
Strains, plasmids and oligonucleotides.
| Strain: | Genotype: | Source: |
|---|---|---|
| Vibrio cholerae | ||
| JB58 | 01 El Tor strain N16961 ΔlacZ Streptomycin-resistant | (Bina et al., 2006) |
| JB460 | JB58 ΔtoxT | (Bina et al., 2003) |
| XBV153 | JB58 ΔaphA | This study |
| Escherichia coli | ||
| EC100Dpir+ | F− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ− rpsL (SmR) nupG pir+ | Epicentre |
| SM10 pir+ | thi-1 thr leu tonA lacY supE recA∷RP4-2-4-Tc∷Mu KmR (λ pirR6K) | (Miller and Mekalanos, 1988) |
| Plasmid: | Description: | |
| pBAD24 | Expression plasmid, CbR, pBR322 origin of replication | (Guzman et al., 1995) |
| pEW1 | pXB300-aphA | This study |
| pJB510 | pBAD24∷toxT, CbR | (Bina et al., 2003) |
| pTL61T | Promoter probe vector containing lacZ; CbR | (Linn and St Pierre, 1990) |
| pWM91 | Allelic exchange vector, CbR | (Metcalf and Wanner, 1993) |
| pXB248 | pWM91 containing 1.9 kb aphA deletion construct | This study |
| pXB300 | TetR-regulated expression vector, pBR322 origin of replication, CbR | This study |
| pXB308 | pXB300-∷lacZ, CbR | This study |
| pXB320 | pXB300-∷toxT, CbR | This study |
| pXB324 | pBAD24-∷lacZ, CbR | This study |
| Oligonucleotides: | DNA sequence (5′ – 3′): | |
| toxT-F-BamHI | ATGGATCCTTCAGAGTAGAACGCAATGATTGG | |
| toxT-R-EcoRV | CTGATATCTAGGATCAAGTAAACGTATTCC | |
| aphA-F-SacI | CGGAGCTCTGGATTGAAGACATGTCATTACC | |
| aphA-R-BamHI | CCGGATCCTTTGGCTTGGCTTATGCCATCGC | |
| lacZ-F-SmaI | ATCATCGGAGCTCTCGAGTCAGCCC | |
| lacZ-R-SphI | AAGCATGCGGGGAGGCAGACAAGGTATAG | |
| aphA-F1-BamHI | TTGGATCCGGCAAGCTGCCATTGGGTTCCAGACCCG | |
| aphA-F2 | AACCGGGTACGATGCCGCTTATTACGCTAACCCAGCCGTG | |
| aphA-R1-SacI | TTGAGCTCCCGAGTATCTCAGAAGCGGCGGCGTGTG | |
| aphA-R2 | CGTAATAAGCGGCATCGTACCCGGTTGCATCGCGTGTGCTAAG |