Table 1.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristic(s) or feature(s) or primer sequencea (5′ to 3′) | Reference or source |
|---|---|---|
| Strains | ||
| E. coli | ||
| JM109 | Cloning strain | |
| S17-1 | Conjugation strain | 43 |
| R. eutropha | ||
| H16 | Wild-type strain | DSMZ 428 |
| H16 ΔphaC1 | Chromosomal deletion of phaC1 | 36 |
| H16 ΔphaP5 | Chromosomal deletion of phaP5 | 14 |
| H16 ΔphaM | Chromosomal deletion of phaM | 13 |
| Plasmids | ||
| pBBR1MCS-2 | Broad-host-range vector, Kmr | 22 |
| pCM62 | Broad-host-range vector, Tcr | 23 |
| pDMW7 | Source of araC-PBAD and rrnB | 44 |
| pEYFP-C1 | Source of eyfp-c1 | Clontech |
| pBBR1MCS-2-PphaC-phaM | Constitutive expression of PhaM (phaC promoter) | 16 |
| pBBR1MCS-2-PphaC-eyfp-phaM | Constitutive expression of eYfp-PhaM fusion (phaC promoter) | 13 |
| pBBR-EYFP-C1 | Plasmid coding for eYfp under the control of the PBAD promoter based on pBBR1MCS-2 | This study |
| pBBR-YN-C1 | Plasmid coding for the N-terminal part of eYfp (YN, amino acids 1 to 154) under the control of the PBAD promoter based on pBBR1MCS-2 | This study |
| pBBR-YC-C1 | Plasmid coding for the C-terminal part of eYfp (YC, amino acids 155 to 238) under the control of the PBAD promoter based on pBBR1MCS-2 | This study |
| pBBR-YN-phaC1 | phaC1 ligated into the XhoI and SpeI sites of pBBR-YN-C1 | This study |
| pBBR-YN-phaP5 | phaP5 ligated into the XhoI and KpnI sites of pBBR-YN-C1 | This study |
| pBBR-YN-phaM | phaM ligated into the KpnI and SpeI sites of pBBR-YN-C1 | This study |
| pCM-EYFP-C1 | Plasmid coding for eYfp under the control of the PBAD promoter based on pCM62 | This study |
| pCM-YC-C1 | Plasmid coding for the C-terminal part of eYfp (YC, amino acids 155 to 238) under the control of the PBAD promoter based on pCM62 | This study |
| pCM-YC-phaC1 | phaC1 ligated into the XhoI and SpeI sites of pCM-YC-C1 | This study |
| pCM-YC-phaP5 | phaP5 ligated into the XhoI and KpnI sites of pCM-YC-C1 | This study |
| pCM-YC-phaM | phaM ligated into the KpnI and SpeI sites of pCM-YC-C1 | This study |
| pJOE4036.1 | Rhamnose-inducible His tag expression vector, Ampr | J. Altenbuchner |
| pJOE4036.1-phaP5(His)6 | Expression vector for PhaP5 | This study |
| pJOE4036.1-phaM(His)6 | Expression vector for PhaM | 13 |
| Primers | ||
| rrnB_fwd_NsiI | CGGCATGCATTGAGTTTAAACGGTCTCCAGCTTGG | |
| rrnB_rev_NsiI | CGGCATGCATGTAGAAACGCAAAAAGGCCATCCG | |
| EYFP-C1_f_NdeI | GGGAATTCCATATGGTGAGCAAGGGCGAGG | |
| EYFP-C1_r_SpeI | GGACTAGTTGATCAGTTATCTAGATCCGGTGGATCC | |
| YC-C1_f_NdeI | GGGAATTCCATATGGCCGACAAGCAGAAGAACGGC | |
| YN-C1_r_XhoI | CCGCTCGAGATCTGAGTCCGGACATGATATAGACGTTGTGGCTGTTGTAG | |
| AdapA | CATGTGCATGCATATGGTTTAAACAGTACTAGTG | |
| AdapB | AATTCACTAGTACTGTTTAAACCATATGCATGCA | |
| PhaC1_C_f_XhoI | CCGCTCGAGGCATGGCGACCGGCAAAGGCGCGG | |
| PhaC1_C_r_SpeI | GGACTAGTTCATGCCTTGGCTTTGACGTATCGCCCAGGC | |
| PhaP5_C_f_XhoI | CCGCTCGAGGCATGGCCACGCCTCCC | |
| PhaP5_C_r_KpnI | GGGGTACCCTAGCCCTTGGATTTCGGCTTG | |
| PhaM_C_f_XhoI | CCGCTCGAGGCATGTTCGGACAGATTCCCG | |
| PhaM_C_r_KpnI | GGGGTACCTCAGGCTGCGCTGCTG | |
| PhaM_C_f_KpnI | GGGGTACCATGTTCGGACAGATTCCCGATTTCACC | |
| PhaM_C_r_SpeI | GGACTAGTTCAGGCTGCGCTGCTGCC |
a
Restriction sites are underlined. Start and stop codons are marked in bold type.