TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| E. coli strain, plasmid, or oligonucleotide | Genotype, description, or sequence | Reference or source |
|---|---|---|
| Strains | ||
| XL1-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 (rK− mK+) supE44 relA1 lac[F′ proAB lacIqlacZΔM15 Tn10 (Tcr)] | 9 |
| BL21(DE3) | F−ompT hsdSB (rB− mB−) gal dcm (DE3) | Novagen |
| Plasmids | ||
| pMCS69 | pBBR1MCS derivative containing the genes phaA and phaB of Ralstonia eutropha colinear to the lac promoter | 17 |
| pET-14b | Apr; T7 promoter | Novagen |
| pETC | pET-14b derivative coding for the phaC wild type under T7 promoter control | 27 |
| pCWE | pBluescript SK(−) derivative containing the NdeI/BamHI-inserted phaC gene from R. eutropha | 26 |
| pMAL-c2G | Plasmid for expression and purification of maltose binding protein fusions | New England Biolabs |
| pCWESpeGFP | pCWESpe containing the SpeI-inserted gfp gene | 25 |
| pET-14b ZZ(−)phaC | pET-14b containing the XbaI/BamHI fragment comprising the ZZ-phaC gene | 8 |
| pET-14b PhaC-linker-MalE | pET-14b derivative containing malE fused to the 3′ end of phaC via a linker sequence | This study |
| pET-14b PhaC-MalE | pET-14b derivative containing malE directly fused to the 3′ end of phaC | This study |
| pET-14b M-PhaC-linker-MalE | pET-14b PhaC-linker-MalE derivative containing the mpl sequence fused to the 5′ end of phaC | This study |
| pET-14b M-PhaC-MalE | pET-14b PhaC-MalE derivative containing the mpl sequence fused to the 5′ end of phaC | This study |
| pMAL-Mpl-EC | pMAL-c2x derivative containing the mpl sequence | 36 |
| pCWESpeMpl-EC | pCWE derivative containing the mpl sequence fused to the 5′ end of phaC | This study |
| pET-14b M-PhaC | pETC derivative containing the mpl sequence fused to the 5′ end of phaC | This study |
| pET-14b PhaC-linker-SG-linker-MalE | pET-14b PhaC-linker-MalE derivative containing the SG linker sequence upstream of malE | This study |
| pET-14b PhaC-linker-GFP | pET-14b derivative containing gfp fused to the 3′ end of phaC via a linker sequence | This study |
| pET-14b PhaC-linker-SG-linker-GFP | pET-14b PhaC-linker-GFP derivative containing the SG linker sequence upstream of gfp | This study |
| pET-14b PhaC-GFP | pET-14b derivative containing gfp directly fused to the 3′ end of phaC | This study |
| pET-14b ZZ-PhaC-GFP | pET-14b PhaC-GFP derivative containing the ZZ sequence 5′ of phaC | This study |
| Oligonucleotides | ||
| linker I | 5′-P-TAT GGT GCT GGC GGT GGC GAT TGA TAA ACG CGG AGG CGG TGG AGG CC-3′ | This study |
| linker II | 5′-P-TCG AGG CCT CCA CCG CCT CCG CGT TTA TCA ATC GCC ACC GCC AGC ACC A-3′ | This study |
| MalE XhoI | 5′-CTC GAG ATG AAA ATC GAA GAA GGT AAA CTG GTA ATC-3′ | This study |
| MalE BamHI | 5′-GGA TCC TTA CTT GGT GAT ACG AGT CTG CGC GTC TTT CAG GGC TTC ATC GAC-3′ | This study |
| PhaC XbaI | 5′-TCT AGA AAT AAG GAG ATA CTA GTA TGG CGA CCG GCA AAG GCG CGG CAG CTT CCA CGC AG-3′ | This study |
| PhaC no stop | 5′-CAT ATG TGC CTT GGC TTT GAC GTA TCG CCC AGG-3′ | This study |
| GFP no start XhoI | 5′-ATG ACC TCG AGA GTA AAG GAG AAG AAC TTT TCA CTG GAG TTG TC-3′ | This study |
| GFP stop BamHI | 5′-GGA TCC TCA TTT GTA TAG TTC ATC CAT GCC ATG TGT AAT-3′ | This study |
| SG linker I | 5′-P-TCG AGA GCG GCG GTG GCG GTA GCG GTG GCG GTG GCA GCG GCG GTG GCG GTA GCC CCG GGC-3′ | 12 (modified) |
| SG linker II | 5′-TCG AGC CCG GGG CTA CCG CCA CCG CCG CTG CCA CCG CCA CCG CTA CCG CCA CCG CCG CTC-3′ | 12 (modified) |
| AscI PhaC | 5′-GGC GCG CCG TGC GCG CTG CT-3′ | This study |
| XhoI PhaC no stop | 5′-CTC GAG TGC CTT GGC TTT GAC GTA T-3′ | This study |