TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in the study
| Strain, plasmid, or oligonucleotide | Relevant characteristicsa | Reference or source |
|---|---|---|
| E. coli strains | ||
| K1060b | F− fadE62 lacI60 tyrT58(AS) fabB5 mel-1 | 69 |
| K1060C | Same as K1060, but creC510 by K1060 × P1(58-161, F− Strr) | This work |
| DB1060 | Same as K1060, but ΔcreB by allelic replacement | This work |
| DC1060 | Same as K1060, but ΔcreC by allelic replacement | This work |
| DBC1060 | Same as K1060, but ΔcreB ΔcreC by allelic replacement | This work |
| CE1060 | Same as DC1060, but ΔadhE by allelic replacement | This work |
| CEA1060 | Same as CE1060, but ΔackA by allelic replacement | This work |
| 58-161, F− Strrb | F− relA1 rpsL100(Strr) spoT1 metB1 creC510 | 70 |
| 58KF | Same as 58-161, F− Strr, but Kmr by insertion of kan in an intergenic region between yjjX and trpR | This work |
| Plasmids | ||
| pCP20 | Helper plasmid used for kan excision; Saccharomyces cerevisiae FLP λ cI857 λPR repA(Ts); Apr Cmr | 25 |
| pKD4 | Template plasmid carrying the FRT-kan-FRT cassette; Kmr | 24 |
| pKD46 | Helper plasmid expressing the λ-Red functions; Apr | 24 |
| pSBF2 | Plasmid pDHK30 (71) carrying FDH1 from Candida boidinii under the control of the lac promoter; Kmr | 43 |
| pEcPpc | Plasmid pTrc99A (72) carrying ppc from E. coli under the control of the lac promoter; Apr | 39 |
| Oligonucleotidesc (5′→3′) | ||
| cre/F | TAG GCC TGA TAA GAC GTG GCG CAT CAG GCA TCG TGC ACC GAA TGC CGG ATG TGT AGG CTG GAG CTG CTT C (K1060C construction) | This work |
| cre/R | GCC GCG TCT TAT CAT GCC TAC CAA ACA TAT TGA AAT TAC GGG TAT TTG TAC ATA TGA ATA TCC TCC TTA G (K1060C construction) | This work |
| creB-KF/F | TTA GCG CGG TTC CTG TCA TGC CGT GGC GGC AAT AAC AGA GGC GAT TTA TGG TGT AGG CTG GAG CTG CTT C (DB1060 and DBC1060 construction) | This work |
| creB-KF/R | GCC CAG CAA CAA CCG CAT GCC GAT ACG CAT TAC AGG CCC CTC AGG CTA TAC ATA TGA ATA TCC TCC TTA (DB1060 construction) | This work |
| creC-KF/F | GTC AAA GAA GTT AAA CCG GGC GTG CGA AGA GCA ACG GAG GGG ACG TTG ATC GTG TAG GCT GGA GCT GCT TC (DC1060 construction) | This work |
| creC-KF/R | GAC GTG TTC CTG ATC CAC TTC GGC GCT TAG CGT GAT GCA ACC GCT CTC GGG CAT ATG AAT ATC CTC CTT AG (DC1060 and DBC1060 construction) | This work |
| DW-ack/F | AAC TCA GCG GGA CAA CG (CEA1060 construction) | This work |
| DW-ack/R | GAA AGC AGA CCT TCA ACG (CEA1060 construction) | This work |
| DW-adh/F | AGA CGC GCT GAC AAT ACG (CE1060 and CEA1060 construction) | This work |
| DW-adh/R | GCC ACC AGA CGC ATA ACC (CE1060 and CEA1060 construction) | This work |
a
Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; Str, streptomycin.
b
Strain obtained through the E. coli Genetic Stock Center, Yale University, New Haven, CT.
c
The use of each oligonucleotide in the construction of mutant strains is indicated in parentheses.