TABLE 1.
E. coli strains, plasmids, and oligonucleotide primers used
| Strain, plasmid, or primer | Relevant genotype or primer sequence (5′→3′)a | Reference or source |
|---|---|---|
| Strains | ||
| DH10B | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139Δ(ara leu)7697 galU galK λ−rpsL nupG | Invitrogen |
| ElectroTen-Blue | Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Kanr [F′ proAB lacIqZΔM15 Tn10 (Tetr)] | Stratagene |
| MG1655 | F− λ−ilvG rfb-50 rph-1 | ATCC 700926 |
| BL21Star(DE3) | F−ompT hsdSB (rB− mB−) gal dcm rne131 (DE3) | Invitrogen |
| MG1655(DE3) | F− λ−ilvG rfb-50 rph-1 (DE3) | This study |
| Plasmids | ||
| pGEM-T Easy | PCR cloning vector; F1 ori Ampr | Promega |
| pETDuet-1 | ColE1(pBR322) ori lacI T7lac Ampr | Novagen |
| pCDFDuet-1 | CloDF13 ori lacI T7lac Strr | Novagen |
| pET-H | pETDuet-1 harboring hbd from C. acetobutylicum ATCC 824 | This study |
| pET-H-Bb | pETDuet-1 harboring bktB from R. eutropha H16 and hbd from C. acetobutylicum ATCC 824 | This study |
| pET-H-Tb | pETDuet-1 harboring thl and hbd from C. acetobutylicum ATCC 824 | This study |
| pET-H-Pb | pETDuet-1 harboring phaA from R. eutropha H16 and hbd from C. acetobutylicum ATCC 824 | This study |
| pET-P | pETDuet-1 harboring phaB from R. eutropha H16 | This study |
| pET-P-Bb | pETDuet-1 harboring bktB and phaB from R. eutropha H16 | This study |
| pET-P-Tb | pETDuet-1 harboring thl from C. acetobutylicum ATCC 824 and phaB from R. eutropha H16 | This study |
| pET-P-Pb | pETDuet-1 harboring phaA and phaB from R. eutropha H16 | This study |
| pCDF-T | pCDFDuet-1 harboring tesB from E. coli MG1655 | This study |
| pCDF-PB | pCDFDuet-1 harboring ptb-buk operon from C. acetobutylicum ATCC 824 | This study |
| Primersc | ||
| bktB_US_EcoRI | GAATTCATGACGCGTGAAGTGGTAGTG | Sigma-Genosys |
| bktB_DS_XhoI | CTCGAGCGCAAGGCTAACCTCAGAT | Sigma-Genosys |
| thil_US_EcoRI | ATAGAATTCCATGAGAGATGTAGTAATAGTAAGTG | Sigma-Genosys |
| thil_DS_XhoI | TATTGAACCTCCTCGAGAACTTAGTTATAT | Sigma-Genosys |
| phaA_US_EcoRI | GAATTCGACTACACAATGACTGACGTTGTC | Sigma-Genosys |
| phaA_DS_XhoI | CTCGAGAAAACCCCTTCCTTATTTGC | Sigma-Genosys |
| hbd_US_EcoRI | GAATTCGGGAGGTCTGTTTAATGAAAA | Sigma-Genosys |
| hbd_DS_NotI | GCGGCCGCTGTAAACTTATTTTG | Sigma-Genosys |
| phaB_US_EcoRI | GAATTCAACGAAGCCAATCAAGGAG | Sigma-Genosys |
| phaB_DS_NotI | GCGGCCGCGCAGGTCAGCCCATATG | Sigma-Genosys |
| tesB_US_EcoRI | GAATTCTACTGGAGAGTTATATGAGTCAGG | Sigma-Genosys |
| tesB_DS_tSalI | GTCGACTTAATTGTGATTACGCATC | Sigma-Genosys |
a
Restriction enzyme sites used in the cloning are underlined.
b
Each gene is under the control of the T7lac promoter with a ribosome binding site.
c
Primers were synthesized at Sigma-Genosys, St. Louis, MO.