TABLE 3.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristicsa | Reference or source and strain collection no. |
|---|---|---|
| Strains | ||
| Escherichia coli BL21(DE3)/pLys | For protein expression | Novagen, 4512 |
| E. coli S17-1 (SN490) | For conjugation | 44 |
| E. coli XL1-Blue | For site-directed mutagenesis | Stratagene, 4505 |
| Ralstonia eutropha H16 (SN1342) | R. eutropha wild-type strain | DSMZ 428 |
| R. eutropha H16 ΔphaC1 (SN6525) | R. eutropha H16 with knockout of phaC1 | 16 |
| R. eutropha H16 ΔphaC1 ΔphaM (SN6506) | R. eutropha H16 with knockout of phaC1 and phaM | This study |
| R. eutropha H16 ΔphaZa1 (SN6167) | R. eutropha H16 with knockout of phaZa1 | This study |
| Plasmids | ||
| pBBR1MCS2 | Broad host range vector; Kmr | 44 |
| pBBR1MCS2-PphaC_eyfp-C1 | Universal vector for construction of fusions C-terminal to eyfp under the control of the phaCAB promoter | 29 |
| pBBR1MCS2_PphaC_phaC1_eyfp | N-terminal fusion of PhaC1 to eYFP | 16 |
| pBBR1MCS2_PphaC_phaC1_T30A_eyfp | N-terminal fusion of PhaC1 (T30A) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T30D_eyfp | N-terminal fusion of PhaC1 (T30D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T373A_eyfp | N-terminal fusion of PhaC1 (T373A) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T373D_eyfp | N-terminal fusion of PhaC1 (T373D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T373N_eyfp | N-terminal fusion of PhaC1 (T373N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T373E_eyfp | N-terminal fusion of PhaC1 (T373E) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T94N_eyfp | N-terminal fusion of PhaC1 (T94N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T94D_eyfp | N-terminal fusion of PhaC1 (T94D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T109N_eyfp | N-terminal fusion of PhaC1 (T109N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T109D_eyfp | N-terminal fusion of PhaC1 (T109D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T94N_T373N_eyfp | N-terminal fusion of PhaC1 (T94N, T373N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T94N_T109N_T373N_eyfp | N-terminal fusion of PhaC1 (T94N, T109N, T373N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T94D_T373D_eyfp | N-terminal fusion of PhaC1 (T94D, T373D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T94D_T109D_T373D_eyfp | N-terminal fusion of PhaC1 (T94D, T109D, T373D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T30D_T94D_T109D_T373D_eyfp | N-terminal fusion of PhaC1 (T30D, T94D, T109D, T373D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaC1_T30N_T94N_T109N_T373N_eyfp | N-terminal fusion of PhaC1 (T30N, T94N, T109N, T373N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaZa1_eyfp | N-terminal fusion of PhaZa1 to eYFP | This study |
| pBBR1MCS2_PphaC_phaZa1_S35D_eyfp | N-terminal fusion of PhaZa1 (S35D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaZa1_S35N_eyfp | N-terminal fusion of PhaZa1 (S35N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaZa1_T26D_eyfp | N-terminal fusion of PhaZa1 (T26D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaZa1_T26N_eyfp | N-terminal fusion of PhaZa1 (T26N) to eYFP | This study |
| pBBR1MCS2_PphaC_phaZa1_T28D_eyfp | N-terminal fusion of PhaZa1 (T28D) to eYFP | This study |
| pBBR1MCS2_PphaC_phaZa1_T28N_eyfp | N-terminal fusion of PhaZa1 (T28N) to eYFP | This study |
| Fusions to His6 tag | ||
| pET28a | His tag expression vector; Kmr | Novagen |
| pET28a_phaC1 | Plasmid for expression of His6-PhaC1 | 20 |
| pET28a_phaC1 (T30A) | Plasmid for expression of His6-PhaC1 (T30A) | This study |
| pET28a_phaC1 (T30D) | Plasmid for expression of His6-PhaC1 (T30D) | This study |
| pET28a_phaC1 (T373A) | Plasmid for expression of His6-PhaC1 (T373A) | This study |
| pET28a_phaC1 (T373D) | Plasmid for expression of His6-PhaC1 (T373D) | This study |
| pET28a_phaC1 (T373E) | Plasmid for expression of His6-PhaC1 (T373E) | This study |
| pET28a_phaC1 (T373N) | Plasmid for expression of His6-PhaC1 (T373N) | This study |
| pET28a_phaM | Plasmid for expression of His6-PhaM | 22 |
| Knockout | ||
| pLO3_phaZa1 | Plasmid used to generate phaZa1 knockout, Tcr | 30 |
a
Kmr, kanamycin resistance; Tcr, tetracycline resistance.