Table 1.
Bacterial strains and plasmids used in this study. CmR and KmR indicate chloramphenicol and kanamycin resistance, respectively. The aa indicates amino acids.
| Bacterial Strains/Plasmids Description | Source | |
|---|---|---|
| Streptomyces sp. SCSIO 02999 | ||
| wild-type | A marine-derived Streptomyces sp., cultured in AM6 medium | [37] |
| E. coli K-12 BW25113 strains | ||
| wild-type | lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78 rph-1 | [68] |
| Δlon | Δlon Δ kmR | [68] |
| BL21(DE3) | F−ompT hsdSB(rB−mB−) gal dcm λ(DE3) Ω PtacUV5::T7 polymerase | Novagen |
| WM3064 | thrB1004 pro thi rpsL hsdS lacZΔM15 RP4-1360) Δ(araBAD)567 ΔdapA1341::[erm pir(wt)] | Metcalf, W.; UIUC |
| Plasmids | ||
| pCA24N | CmR; lacIq | [69] |
| pCA24N-vapB | CmR; lacIq, PT5-lac::vapB | this study |
| pCA24N-vapC | CmR; lacIq, PT5-lac::vapC | this study |
| pCA24N-vapB-vapC | CmR; lacIq, PT5-lac::vapB-vapC | this study |
| pCA24N-vapCD6A | CmR; lacIq, PT5-lac::vapCD5A, mutant the 5th aa of VapC from D to A | this study |
| pCA24N-vapCD96A | CmR; lacIq, PT5-lac::vapC, mutant the 95th aa of VapC from D to A | this study |
| pCA24N-vapCD114A | CmR; lacIq, PT5-lac::vapC, mutant the 113th aa of VapC from D to A | this study |
| pET28b | KmR, lacIq | - |
| pET28b-vapB-vapC-CHis | KmR, lacIq, pET28b PT7-lac:: vapB-vapC with VapC C-terminal His-tagged | this study |
| pET28b-vapB-vapC | KmR, lacIq, pET28b PT7-lac:: vapB-vapC without His-tag | this study |
| pHGEI01 | KmR, R6K ori, pHGC01 containing the full-length E. coli lacZ gene | [70] |
| pHGEI01-vapB’ | pHGEI01 containing the Streptomyces sp. native promoter and inactive VapB N-termianl 15aa | this study |
| pHGEI01-vapC’ | pHGEI01 containing the Streptomyces sp. native promoter and VapB inactive VapC N-termianl 15aa | this study |
| pHGEI01-vapB-vapC | pHGEI01 containing the Streptomyces sp. native promoter and vapB and vapC | this study |
R: resistance, q: quantity, and UIUC standards for University of Illinois at Urbana-Champaign.4.2. Cloning of Genes