TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Description | Reference or source |
|---|---|---|
| Strains | ||
| Gordonia sp. | ||
| SoCg | Long-chain n-alkane degrader, alkB+ | 16 |
| SoCg ΩalkB | SoCg disruption mutant, alkB− Aprar | This study |
| Escherichia coli | ||
| DH10B | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74recA1 endA1 araD139 Δ(ara leu)7697 galU galK λ−rpsL nupG | Invitrogen |
| ET12567 | F−dam-13::Tn9 dcm-6hsdM hsdRzjj-202::Tn10recF143galK2galT22ara14 lacY1 xyl-5 leuB6 Cmlr Tetr Kanr | 9 |
| BL21(DE3)/pLysS | F−ompT hsdSB (rB− mB−) dcmgal λ(DE3) pLysS Camr | Invitrogen |
| BL21-AH | E. coli BL21 containing the recombinant expression vector pRalkB; alkB Ampr | This study |
| Streptomyces coelicolor | ||
| M145 | Wild type; SCP1− SCP2− | 9 |
| M145-AH | S. coelicolor M145 containing the recombinant integrative pIalkB; alkB Thior Aprar | This study |
| Cloning and expression vectors | ||
| pUC18 | E. coli cloning vector, Ampr | Invitrogen |
| pGEM-T Easy vector | E. coli cloning vector, Ampr | Promega |
| TOPO-TA | E. coli cloning vector, Ampr | Invitrogen |
| pRSET-B | E. coli expression vector, Ampr | Invitrogen |
| pIJ8600 | Streptomycete expression vector, Aprar, promoter induction by thiostrepton | 9 |
| pIJ773 | Streptomycete cloning vector, Aprar, used to extract apramycin resistance cassette aac(3)IV with its oriT | 9 |
| Plasmids containing DNA from Gordonia SoCg | ||
| palkCg23 | pGEM-T Easy vector derivative containing a 570-bp alkB fragment (GenBank accession no. EF437969) | 16 |
| palk68 | pUC18 derivative containing an 8-kb fragment of Gordonia SoCg including the alk cluster | This study |
| pGalkB1 | pGEM-T Easy vector derivative containing alkB (1.2 kb), amplified by PCR with primers alkNIFor and alkBHIRev | This study |
| pRalkB | pRSET-B derivative containing alkB (1.2 kb) | This study |
| pIalkB | pIJ8600 derivative containing alkB (1.2 kb) | This study |
| palkapra | palk68 derivative containing the apramycin resistance cassette cloned into a unique AleI restriction site of alkB | This study |