TABLE 1.
Bacterial strains, plasmids, and oligonucleotides
| Strain, plasmid, or oligonucleotide | Description or sequencea | Source, reference, or location |
|---|---|---|
| Strains | ||
| R. eutropha H16 | Wild type; Gmr | DSM 428 |
| E. coli TOP10 | Cloning strain; F− mcrA Δ(mrr-hsdRMS-mcrBC) galK rpsL ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU (Strr) endA1 nupG | Life Technologies |
| E. coli BL21(DE3) | Expression strain; E. coli B F− dcm ompT hsdS(rB− mB−) gal (DE3) | Stratagene |
| Plasmids | ||
| pCR2.1-TOPO | Cloning vector; Apr Kmr | Life Technologies |
| pBBR1MCS-2 | Broad-host-range vector; Kmr lacPOZ′ mobRP4 | Life Technologies |
| pETDuet-1 | Expression vector with two MCSs, each preceded by a T7 promoter/lac operator and rbs; ColE1 ori lacI Apr | Merck Millipore |
| pBHR68 | pBluescriptSK(−) harboring the phaCABRe operon including the promoter; Apr | 50 |
| pBBR1MCS-2::phaCAB | pBBR1MCS-2 carrying the BamHI-EcoRI fragment of pBHR68 harboring the phaCAB operon including the promoter; Kmr | 35 |
| pETDuet-1::phaP1 | pETDuet carrying phaP1Re in MCS-1; Apr | 35 |
| pETDuet-1::phaP2 | pETDuet carrying phaP2Re in MCS-1; Apr | 35 |
| pETDuet-1::phaP4 | pETDuet carrying phaP4Re in MCS-1; Apr | 35 |
| pETDuet-1::phaZ1 | pETDuet carrying phaZ1Re in MCS-2; Apr | 35 |
| pETDuet-1::phaZ2 | pETDuet carrying phaZ2Re in MCS-2; Apr | This study |
| pETDuet-1::phaZ3 | pETDuet carrying phaZ3Re in MCS-2; Apr | This study |
| pETDuet-1::phaZ7 | pETDuet carrying phaZ7Re in MCS-2; Apr | This study |
| pETDuet-1::phaP1::phaZ1 | pETDuet carrying phaP1Re in MCS-1 and phaZ1Re in MCS-2; Apr | 35 |
| pETDuet-1::phaP1::phaZ2 | pETDuet carrying phaP1Re in MCS-1 and phaZ2Re in MCS-2; Apr | This study |
| pETDuet-1::phaP1::phaZ3 | pETDuet carrying phaP1Re in MCS-1 and phaZ3Re in MCS-2; Apr | This study |
| pETDuet-1::phaP1::phaZ7 | pETDuet carrying phaP1Re in MCS-1 and phaZ7Re in MCS-2; Apr | This study |
| pETDuet-1::phaP2::phaZ1 | pETDuet carrying phaP2Re in MCS-1 and phaZ1Re in MCS-2; Apr | 35 |
| pETDuet-1::phaP2::phaZ2 | pETDuet carrying phaP2Re in MCS-1 and phaZ2Re in MCS-2; Apr | This study |
| pETDuet-1::phaP2::phaZ3 | pETDuet carrying phaP2Re in MCS-1 and phaZ3Re in MCS-2; Apr | This study |
| pETDuet-1::phaP2::phaZ7 | pETDuet carrying phaP2Re in MCS-1 and phaZ7Re in MCS-2; Apr | This study |
| pETDuet-1::phaP4::phaZ1 | pETDuet carrying phaP4Re in MCS-1 and phaZ1Re in MCS-2; Apr | 35 |
| pETDuet-1::phaP4::phaZ2 | pETDuet carrying phaP4Re in MCS-1 and phaZ2Re in MCS-2; Apr | This study |
| pETDuet-1::phaP4::phaZ3 | pETDuet carrying phaP4Re in MCS-1 and phaZ3Re in MCS-2; Apr | This study |
| pETDuet-1::phaP4::phaZ7 | pETDuet carrying phaP4Re in MCS-1 and phaZ7Re in MCS-2; Apr | This study |
| Oligonucleotides | ||
| phaZ2_NdeI | 5′-GCGGGGACCGAGCCATATGCTGTACCAC-3′ | 5′ region of phaZ2 |
| phaZ2_KpnI | 5′-GCGCGCTGGCGGGTACCGGGTTAGCTGCTGG-3′ | 3′ region of phaZ2 |
| phaZ3_NdeI | 5′-GGCGGGAATCGCACATATGGCGCTCTATGCTCTGCG-3′ | 5′ region of phaZ3 |
| phaZ3_KpnI | 5′-CCTCGCCAAGGCGACGGAGCGCGGTACCTCAGTCGGCGCACC-3′ | 3′ region of phaZ3 |
| phaZ7_NdeI | 5′-GACAGGACGCCATATGCCCCGCTCATC-3′ | 5′ region of phaZ7 |
| phaZ7_KpnI | 5′-CAAGCCTGCCGGTACCTCACCCCCCAAC-3′ | 3′ region of phaZ7 |
a
phaCABRe, R. eutropha phaCAB.