Table 1 Bacterial strains, plasmids, and primers used in this study
Strain name | Genotype, description, or sequence | Source/References |
|---|---|---|
E. coli DH5α | F−, φ80dlacZ ΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rK−, mK+), phoA, supE44, λ−, thi-1, gyrA96, relA1 | Takara |
E. coli BL21(DE3) | F−ompT hsdSB(rB− mB−)gal dcm (DE3) | TIANGEN |
Plasmids | ||
pET22b (+)-cgt | Ampicillin resistance, pET22b ( +) derivative containing the cgt gene, to produce free γ-CGTase | Duan et al. [24] |
pCDFDuet-1 | Streptomycin resistance, dual T7 promoter | Novagen |
pCDFD-AB | pCDFDuet-1 derivative containing the phaAB genes from Ralstonia eutropha, phaAB genes are under the control of the first T7 promoter | Ran et al. [32] |
pCDFD-ABC | pCDFDuet-1 derivative containing the phaABC operon from Ralstonia eutropha, to produce PHA blank nano-granules. The phaAB genes are under the control of the first T7 promoter and the phaC gene is under the control of the second T7 promoter | Ran et al. [32] |
pCDFD-ABC-cgt | pCDFDuet-1 derivative containing the phaAB genes under the control of the first T7 promoter and the cgt gene from Bacillus clarkii 7364 fused to the 5′ end of phaC from Ralstonia eutropha via a linker sequence under the control of the second T7 promoter, to produce PHA nano-granules decorated with γ-CGTase on the surface | This study |
Primers | ||
phaC XhoI | 5′-AAACTCGAGTGATGGCGACCGGCAAAGGC-3′ | This study |
phaC no start XhoI | 5′-AAACTCGAGGCGACCGGCAAAGGCG-3′ | This study |
phaC stop AvrII | 5′-TTTCCTAGGTCATGCCTTGGCTTTGACGTATCG-3′ | This study |
γ-CGTase BglII | 5′-GAAGATCTAGCAAGTAATGCAACGAACG-3′ | This study |
γ-CGTase XhoI | 5′-CTCGAGGGAGCCACCTCCCGATCCGCCACCAGAGCCACCTCCTCTTCGAAAACTTACAC-3′ | This study |