Table 1. The strains, plasmids, and oligonucleotides used in this study.
| Description | Contents | Reference/sources |
|---|---|---|
| Strain | ||
| E. coli Bl21star(DE3) | B F− ompT gal dcm lon hsdSB(rB−mB−) rne131 (DE3) | Invitrogen |
| E. coli TOP10 | F– mcrA crmrr-hsdRMS-mcrBC) Φ80lacZac80 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG | Invitrogen |
| Plasmid | ||
| pET28a | Invitrogen | |
| pET28a-6pgdh | Precusor of pET28a-Ptac-6pgdh | in this study |
| pET28a-Ptac-6pgdh | dual promoter (PT7and Ptac) and moth6pgdh | In this study |
| primers* | ||
| 28_back_F | 5′-CGACCAAGACCGACTAAGCCGATATGCATATGTATATCTCCTTCTTAAAG-3′ | pET28a |
| 28_back_R | 5′-GGCCACGACGTGGCCCGGAAACACCACCACCACCACCACTGAGAT-3′ | |
| 6PG_F | 5′-CTTTAAGAAGGAGATATACATATGCATATCGGCTTAGTCGGTCTTGGTCG-3′ | Moorella thermoacetica |
| 6PG_R | 5′-ATCTCAGTGGTGGTGGTGGTGGTGTTTCCGGGCCACGTCGTGGCC-3′ | |
| T7_Tac_F | 5′-Phos-GGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTC-3′ | pET28a-6pgdh |
| T7_Tac_R | 5′-Phos-GATGATTAATTGTCAACCTATAGTGAGTCGTATTAATTTCG-3′ | |
| 31_NNK_F | 5′-GAAGTGCGAGGATACGCCNNKACTAAGGCTACCGTGG-3′ | pET28a-Ptac-6pgdh |
| 31_NNK_R | 5′-CCACGGTAGCCTTAGTMNNGGCGTATCCTCGCACTTC-3′ | |
| 30_32_NNK_F | 5′-CATGGTCATGAAGTGCGAGGATACNNKATTNNKAAGGCTACCGTGGACAAAGC-3′ | pET28a-Ptac-6pgdh R31I |
| 30_32_NNK_R | 5′-GCTTTGTCCACGGTAGCCTTMNNAATMNNGTATCCTCGCACTTCATGACCATG-3′ | |
*Boldface nucleotide sequences indicate randomized positions.