TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Descriptiona | Reference or source |
|---|---|---|
| R. eutropha strains | ||
| H16 | Wild-type strain; Gm resistant | ATCC 17699 |
| Re2106 | ΔphaB2; made from H16 with pCB28 | This study |
| Re2107 | ΔphaB3; made from H16 with pCB29 | This study |
| Re2111 | ΔphaB1; made from H16 with pCB42 | This study |
| Re2112 | ΔphaB1 ΔphaB2; made from Re2106 with pCB42 | This study |
| Re2113 | ΔphaB1 ΔphaB3; made from Re2107 with pCB42 | This study |
| Re2114 | ΔphaB2 ΔphaB3; made from Re2106 with pCB29 | This study |
| Re2115 | ΔphaB1 ΔphaB2 ΔphaB3; made from Re2112 with pCB29 | This study |
| Re2139 | phaB1 inserted into Re2115 genome; made with pCB65 | This study |
| Re2140 | phaB2 inserted into Re2115 genome; made with pCB66 | This study |
| Re2141 | phaB3 inserted into Re2115 genome; made with pCB67 | This study |
| Re2142 | fabG inserted into Re2115 genome; made with pCB68 | This study |
| Re2143 | phaB3 inserted into Re2115 genome; made with pCB76 | This study |
| E. coli strains | ||
| DH5α | General cloning strain | Invitrogen |
| S17-1 | Strain for conjugative transfer of plasmids to R. eutropha | 38 |
| Tuner(DE3) | Strain for inducible protein expression | Novagen |
| Plasmids | ||
| pCR2.1-TOPO | Vector for cloning and sequencing PCR products, confers Km resistance | Invitrogen |
| pGY46 | Plasmid for deletion of R. eutropha phaC1; carries sacB, oriV, oriT, and traJ; confers Km resistance; based on pJQ200mp18; all other deletion plasmids use backbone from this plasmid | 31, 46 |
| pCB28 | Plasmid for deletion of R. eutropha phaB2 | This study |
| pCB29 | Plasmid for deletion of R. eutropha phaB3 | This study |
| pCB42 | Plasmid for deletion of R. eutropha phaB1 | This study |
| pCB65 | Plasmid for insertion of phaB1 into R. eutropha genome at phaB1 locus; based on pCB42 | This study |
| pCB66 | Plasmid for insertion of phaB2 into R. eutropha genome at phaB1 locus; based on pCB42 | This study |
| pCB67 | Plasmid for insertion of phaB3 (annotated start codon) into R. eutropha genome at phaB1 locus; based on pCB42 | This study |
| pCB68 | Plasmid for insertion of fabG into R. eutropha genome at phaB1 locus; based on pCB42 | This study |
| pCB76 | Plasmid for insertion of phaB3 (upstream start codon) into R. eutropha genome at phaB1 locus; based on pCB42 | This study |
| pET-15b | Plasmid expression of His-tagged proteins in E. coli; confers Ap resistance | Novagen |
| pRARE2 | Plasmid supplying tRNAs for seven rare E. coli codons; confers Cm resistance | Novagen |
| pET-15b-phaB1 | Plasmid for expression of PhaB1 with N-terminal His tag | This study |
| pET-15b-phaB2 | Plasmid for expression of PhaB2 with N-terminal His tag | This study |
| pET-15b-phaB3 | Plasmid for expression of PhaB3 with N-terminal His tag | This study |
| pET-15b-fabG | Plasmid for expression of FabG with N-terminal His tag | This study |
a
Abbreviations: Gm, gentamicin; Km, kanamycin; Ap, ampicillin; Cm, chloramphenicol.