TABLE 1.
Bacterial strains, plasmids, and primers used in this study.
| Plasmids, strains and primers | Description | Source | ||
| Plasmids | ||||
| pACYCDuet1 | T7 promoter, lacIq, p15A ori, Cmr | Novagen | ||
| pETDuet1 | T7 promoter, lacIq, pBR322 ori, Ampr | Novagen | ||
| pET-28a | T7 promoter, lacIq, pBR322 ori, Kanr | Novagen | ||
| pACYCDuet-adhE | lac1-6 promoter, adhE, lacIq, p15A ori, Cmr | This study | ||
| pACYCDuet-adhEmut | lac1-6 promoter, adhE A267T/E568K, lacIq, p15A ori, Cmr | This study | ||
| pACYCDuet-adhEmut-dnaKJ | lac1-6 promoter, adhE A267T/E568K, dnaKJ, lacIq, p15A ori, Cmr | This study | ||
| pACYCDuet-adhEmut-POS5 | lac1-6 promoter, adhE A267T/E568K, POS5, lacIq, p15A ori, Cmr | This study | ||
| pACYCDuet-acs | lac1-6 promoter, acs, lacIq, p15A ori, Cmr | This study | ||
| pBAD-Ae-pha | araBAD promoter, phaCAB, Ampr, araC, pBR322 ori | (Aldor and Keasling, 2001) | ||
| pET-MCRCN | pBR322 ori, lacIq, T7 promoter, mcr550–1219 N940V/K1106W/S1114R, lac2–51 promoter, mcr1–549, Ampr | Lab collection | ||
| pET28a-phlD | trc promoter, phlD, lacIq, pBR322 ori, Kanr | Lab collection | ||
| Strains | ||||
| E. coli DH5α | F–, supE44, ΔlacU169, (φ80 lacZΔM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1 | Lab collection | ||
| E. coli W3110 | F–, λ–, IN(rrnD-rrnE)1, rph-1 | Lab collection | ||
| E. coli MG1655 (DE3) | F–, λ(DE3), ilvG–, rfb-50, rph-1 | Lab collection | ||
| E. coli BL21(DE3) | F–, ompT, gal, dcm, lon, hsdSB, (rB–, mB–), λ(DE3) | Lab collection | ||
| S. cerevisiae CEN.PK2-1C | MATa; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2 | Lab collection | ||
| Q3352 | E. coli W3110/pACYCDuet1 | This study | ||
| Q3092 | E. coli W3110/pACYCDuet-adhEmut | This study | ||
| Q3094 | E. coli W3110/pACYCDuet-adhEmut/pBAD-Ae-pha | This study | ||
| Q3095 | E. coli W3110/pACYCDuet1/pBAD-Ae-pha | This study | ||
| Q3115 | E. coli BL21(DE3)/pACYCDuet-adhEmut/pBAD-Ae-pha | This study | ||
| Q3133 | E. coli W3110/pACYCDuet-adhEmut-dnaKJ/pBAD-Ae-pha | This study | ||
| Q3135 | E. coli W3110/pACYCDuet-adhEmut-POS5/pBAD-Ae-pha | This study | ||
| Q3140 | E. coli W3110/pACYCDuet-acs/pBAD-Ae-pha | This study | ||
| Q3397 | E. coli MG1655 (DE3)/pACYCDuet1/pET-MCRCN | This study | ||
| Q3398 | E. coli MG1655 (DE3)/pACYCDuet1-adhEmut/pET-MCRCN | This study | ||
| Q3399 | E. coli MG1655 (DE3)/pACYCDuet1-acs/pET-MCRCN | This study | ||
| Q3432 | E. coli W3110/pACYCDuet1/pET28a-phlD | This study | ||
| Q3433 | E. coli W3110/pACYCDuet1-adhEmut/pET28a-phlD | This study | ||
| Q3434 | E. coli W3110/pACYCDuet1-acs/pET28a-phlD | This study | ||
| Primers | Sequence(5′–3′) | Restriction site | ||
| adhE-F | CGCGGATCCGATGGCTGTTACTAATGTCGC | BamHI | ||
| adhE-R | CCGGAGCTCTTAAGCGGATTTTTTCGCTT | SacI | ||
| adhE(A267T)-F | CGCTGTACGTGAACGTTTTACCACCCACGGCGGCTATCTGTT | – | ||
| adhE(A267T)-R | AACAGATAGCCGCCGTGGGTGGTAAAACGTTCACGTACAGCG | – | ||
| adhE(E568K)-F | ATCCGGAAACTCACTTCGAAAAACTGGCGCTGCGCTTTATGGA | – | ||
| adhE(E568K)-R | TCCATAAAGCGCAGCGCCAGTTTTTCGAAGTGAGTTTCCGGAT | – | ||
| acs-F | CGCGGATCCGATGAGCCAAATTCACAAACAC | BamHI | ||
| acs-R | CCGGAGCTCTTACGATGGCATCGCGATAGCC | SacI | ||
| dnaKJ-F | ATAAGAATGCGGCCGCTTACAGACTCACAACCACAT | NotI | ||
| dnaKJ-R | GGAATTCCATATGTTAGCGGGTCAGGTCGTCAA | NdeI | ||
| POS5-F | ATAAGAATGCGGCCGCTTAATAAGGAGATATACCATGAGTACGTTGGATTCACATTCC | NotI | ||
| POS5-R | GGAATTCCATATGTTAATCATTATCAGTCTGTCTCTTGG | NdeI | ||