Table 1.
E. coli strains, plasmids and oligonucleotides used
| Name | Relevant Genotype | Reference |
|---|---|---|
| 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 |
| HCT 10 | MG1655 ΔendA ΔrecA (DE3) | This study |
| HCT 11 | MG1655 ΔendA ΔrecA ΔatoB (DE3) | This study |
| HCT 20 | MG1655 ΔendA ΔackA-pta ΔatoDA ΔpoxB (DE3) | This study |
| HCT 21 | MG1655 ΔendA ΔackA-pta ΔatoDA ΔpoxB ΔmetA Δtdh (DE3) | This study |
| Plasmids | ||
| pETDuet-1 | ColE1(pBR322) ori, lacI, T7lac, AmpR | Novagen |
| pCDFDuet-1 | CloDF13 ori, lacI, T7lac, StrepR | Novagen |
| pCOLADuet-1 | COLA ori, lacI, T7lac, KanR | Novagen |
| pET-B | pETDuet-1 harboring bktB from R. eutropha H16 | This study |
| pET-PB-B a | pETDuet-1 harboring ptb-buk operon from C. acetobutylicum ATCC 824, and bktB from R. eutropha H16 | This study |
| pCDF-H | pCDFDuet-1 harboring hbd from C. acetobutylicum ATCC 824 | This study |
| pCDF-T-H a | pCDFDuet-1 harboring tesB from E. coli MG1655, and hbd from C. acetobutylicum ATCC 824 | This study |
| pCDF-P | pCDFDuet-1 harboring phaB from R. eutropha H16 | This study |
| pCDF-T-P a | pCDFDuet-1 harboring tesB from E. coli MG1655, and phaB from R. eutropha H16 | This study |
| pCOLA-Iec | pCOLADuet-1 harboring ilvA from E. coli MG1655 | This study |
| pCOLA-Icg | pCOLADuet-1 harboring ilvA from C. glutamicum | This study |
| pCOLA-Tec-Icg a | pCOLADuet-1 harboring thrABC operon from E. coli MG1655, and ilvA from C. glutamicum ATCC 13032 | This study |
| pCOLA-Tecm-Icg a | pCOLADuet-1 harboring thrAG1297ABC operon from E. coli ATCC 21277, and ilvA from C. glutamicum ATCC 13032 | This study |
| Primersb | Sequence 5'→3'c | |
| bktB_US_EcoRI | GAATTCATGACGCGTGAAGTGGTAGTG | Sigma-Genosys |
| bktB_DS_XhoI | CTCGAGCGCAAGGCTAACCTCAGAT | Sigma-Genosys |
| hbd_US_NdeI | ATTCATATGAAAAAGGTATGTGTTATAGG | Sigma-Genosys |
| hbd_DS_AvrII | ATTCCTAGGCAGGTCGACTCTAGAACTTA | Sigma-Genosys |
| phaB_US_MfeI | ATTCAATTGACGAAGCCAATCAAGGAG | Sigma-Genosys |
| phaB_DS_AvrII | ATTCCTAGGGGTCAGCCCATATGCAG | Sigma-Genosys |
| tesB_US_NcoI | ATTCCATGGGCATGAGTCAGGCGCTAA | Sigma-Genosys |
| tesB_DS_NotI | ATTGCGGCCGCGACTCTAGAGACTTAATTGTG | Sigma-Genosys |
| ilvAec_US_NdeI | ATTACATATGGCTGACTCGCAAC | Sigma-Genosys |
| ilvAec_DS_AvrII | ATTACCTAGGCATTTTTCCCTAACC | Sigma-Genosys |
| ilvAcg_US_NdeI | ATTACATATGAGTGAAACATACGTGTC | Sigma-Genosys |
| ilvAcg_DS_AvrII | ATTACCTAGGCCTTCAGCTATGTTTA | Sigma-Genosys |
| thrABC_US_BamHI | ATTAGGATCCAAGGAGATATATCATGCGAGTGTTGAAG | Sigma-Genosys |
| thrABC_US_NcoI | ATTACCATGGGCATGCGAGTGTTGAAG | Sigma-Genosys |
| thrABC_DS_SalI | ATTAGTCGACGATAATGAATAGATTTTACTGATG | Sigma-Genosys |
a Each gene is under the control of the T7lac promoter with a ribosome binding site.
b Primers were synthesized at Sigma-Genosys, St. Louis, MO.
c Restriction enzyme sites used in the cloning are shown in underlined italics.