TABLE 1.
Bacterial strains, plasmids, and primers
| Strain, plasmid, or primer | Relevant characteristic(s) or primer sequence (5′-3′) | Reference or source |
|---|---|---|
| Strains | ||
| Streptomyces sp. SN-593 | Wild-type reveromycin A-producing strain | 43 |
| E. coli DH5α | Cloning host | Takara |
| E. coli BL21 Star (DE3) | Host strain for recombinant protein expression | Invitrogen |
| E. coli EPI300-T1 | Host strain for fosmid library construction | Epicentre Biotechnology |
| E. coli BW 25113 | lacIqrrnBT14 ΔlacZWJ16hsdR514 ΔaraBADAH33 ΔrhaBADLD78 (derivative of the F− λ−E. coli K-12 strain BD792 [CGSC6159]) | 12 |
| E.coli conjugation strain | E. coli GM2929 hsdS::Tn10 carrying pUB307-aph::Tn7 | 26 |
| Plasmids | ||
| pUC118 | General cloning vector with multiple cloning sites; 2- to 5-kb DNA fragments from Streptomyces sp. SN-593 were inserted into HincII site | Takara |
| pET28b | T7 RNA polymerase-dependent recombinant protein expression vector; an N-terminal His6 tag allows protein purification by Ni-affinity column chromatography | Takara |
| pET28b(+)-iptA | The iptA fragment (1,158 bp) was inserted into NdeI and XhoI sites of pET28b(+) | This study |
| pCC1FOS | Optimum vector for constructing a fosmid library harboring about 40 kb of DNA fragments | Epicentre Biotechnology |
| pKU250 | E. coli-Streptomyces conjugation vector | 29 |
| pIM | DNA region between BamHI and KpnI sites was removed from pKU250 | This study |
| pIM-ΔiptA | iptA gene disruption plasmid | This study |
| pKD46 | Red recombinase expression plasmid | 12 |
| pKD13 | Template plasmid for FRT-flanked kanamycin resistance gene | 12 |
| Primers | ||
| NdeI-F | GGAATTCCATATGGTGACCGGCGGCCGCTCCCCGAa | This study |
| XhoI-R | CCGCTCGAGTCAGCGTCCGATCCCGGGCCCGGTa | This study |
| iptA-For-P4 | GAGCCGACGCAGCTCGGTGGTCTCGTCACCGACCAGCTCATTCCGGGGATCCGTCGACCb | This study |
| iptA-Rev-P1 | CTGCACCGCGTACGCCTCGGAGGAGACGTAGACGGTGACTGTAGGCTGGAGCTGCTTCb | This study |
| iptA-SalI-F | GTCGACCTGCTCAACGACTACGCC | This study |
| iptA-SalI-R | GTCGACCATCAGCAGGGCCGTACC | This study |
a
Underlining indicates the corresponding restriction enzyme sites.
b
Underlining shows the 39-bp sequence homologous to the target DNA region.