Table 1.
Bacterial strains and plasmids used in this study
| Strain, plasmid, or fosmid | Characteristicsa | Source or reference(s) |
|---|---|---|
| Strains | ||
| Escherichia coli | ||
| DH5α | Plasmids construction and general subcloning, F−recAlacZΔM15 | New England Biolabs |
| ET12567/pUZ8002 | Nonmethylating ET12567 containing nontransmissible RP4 derivative plasmid pUZ8002; Cmlr Tetr Kanr | 21 |
| Streptomyces | ||
| KCTC 11604BP | Wild-type FK506-producing strain | 24 |
| WT/pSET152(PermE*) | Wild-type stain with integrative plasmid pSET152(PermE*) | This study |
| WT/pTCS2 | Wild-type with integrative plasmid pTCS2; Aprar | This study |
| WT/pTCS7 | Wild-type with integrative plasmid pTCS7; Aprar | This study |
| WT/pFKBN | Wild-type with integrative plasmid pFKBN; Aprar | This study |
| Δtcs2 | Mutant of KCTC 11604BP with an in-frame deletion internal to tcs2 | 24 |
| Δtcs7 | Mutant of KCTC 11604BP with an in-frame deletion internal to tcs7 | This study |
| ΔfkbN | Mutant of KCTC 11604BP with an in-frame deletion internal to fkbN | This study |
| Δtcs7/pFKBN | Δtcs7 with integrative plasmid pFKBN; Aprar | This study |
| ΔfkbN/pTCS2 | ΔfkbN with integrative plasmid pTCS2; Aprar | This study |
| ΔfkbN/pTCS7 | ΔfkbN with integrative plasmid pTCS7; Aprar | This study |
| ΔfkbN/pFKBN | ΔfkbN with integrative plasmid pFKBN; Aprar | This study |
| Plasmids | ||
| pCCFOS1(fosmid) | Vector for genomic library construction; F−oriV Cmlr | Epicentre Biotechnol. |
| pGEM-T Easy | E. coli vector for cloning PCR products; Ampr | Promega |
| pSET152(PermE*) | Integrative plasmid containing PermE*, oriT, attP, ΦC31int, and aac(3)IV | 3, 31 |
| pKC1139 | Temperature-sensitive E. coli-Streptomyces shuttle vector containing oriT and aac(3)IV for gene disruption | 3 |
| pTCS2 | pSET152(PermE*)-based integrative plasmid containing single copy of tcs2; Aprar | This study |
| pTCS7 | pSET152(PermE*)-based integrative plasmid containing single copy of tcs7, Aprar | This study |
| pFKBN | pSET152(PermE*)-based integrative plasmid containing single copy of fkbN; Aprar | This study |
| pΔTCS2 | pKC1139 based deletion plasmid with in-frame deletion of 171 bp internal to tsc2 | This study |
| pΔTCS7 | pKC1139-based deletion plasmid with in-frame deletion of 888 bp internal to tsc7 | This study |
| pΔFKBN | pKC1139-based deletion plasmid with in-frame deletion of 2,772 bp internal to fkbN | This study |
| Fosmids | ||
| fos1004F01 | Fosmid clone; contains bases 1-40 and 366 of FK506 biosynthetic gene cluster | 24 |
| fos1006D05 | Fosmid clone; contains bases 58, 172–197, and 743 of FK506 biosynthetic gene cluster | 24 |
a
F−, does not carry the F plasmid; recA1, for reduced occurrence of unwanted recombination in cloned DNA; lacZΔM15, partial deletion of the lacZ gene, which allows α complementation of the β-galactosidase gene; Cmlr, chloramphenicol resistance; Tetr, tetracycline resistance; Kanr, kanamycin resistance; Aprar, apramycin resistance; Ampr, ampicillin resistance; oriV, origin of replication; oriT, origin of transfer; attP, plasmid ΦC31 attachment site; ΦC31 int, integrase gene; aac(3)IV, apramycin resistance gene; PermE*, mutated constitutive promoter.