Table 2.
Strains, plasmids, and oligonucleotides used in this study
| Strains | Relevant characteristics | Reference |
|---|---|---|
|
Strains |
|
|
| BL21-Gold (DE3) |
E. coli B F– ompT hsdS(rB– mB–) dcm+ TetR gal λ(DE3) endA Hte |
Agilent technologies |
| XL1 Blue |
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacIqZΔM15 Tn10 (Tetr)] |
Agilent technologies |
| Cupriavidus necator H16 |
Wild type, PHA producing |
ATCC 17699 |
|
Plasmids |
|
|
| pLG575 |
pACYC184 derivative, HlyBD, p15A origin, CmR |
[47] |
| pBHR68 |
pBluescript SK-, phbCAB genes from R. eutropha |
[16] |
| pSB1AK3 |
High copy BioBrick vector, pMB1 origin, AmpR and KanR |
[49] |
| pSB1A3 |
High copy BioBrick vector, pMB1 origin, AmpR |
[49] |
| pSB3K3 |
Medium copy BioBrick standard vector, p15A origin, KanR |
[49] |
| pCMEL1 |
phaP1, C-terminal BioFusion with HlyA signal peptide, Lac promoter (BBa_R0010), RBS(BBa_B0034), in pSB1A3 |
This study |
| pCMEL2 |
phaP1, C-terminal BioFusion with HlyA signal peptide, Lac promoter (BBa_R0010), RBS(BBa_B0034), in pSB3K3 |
This study |
| pCMEL3 |
phaP1, C-terminal BioFusion with HlyA signal peptide, Lac promoter (BBa_R0010), RBS(BBa_B0034), in pBHR68 |
This study |
|
Oligonucleotides |
|
|
| PhaP1FOR |
5′-gaattcgcggccgcttctagaatgatcctcaccccggaaca-3′ |
This study |
| PhaP1REV |
5′- ctgcagcggccgctactagttcaggcagccgtcgtcttct-3′ |
This study |
| g114t |
5′-cgtcgagctgaaccttcaggtcgtcaagact-3′ |
This study |
| g114t_antisense | 5′-agtcttgacgacctgaaggttcagctcgacg-3′ | This study |