Description | Contents | Reference/sources |
---|---|---|
Strain | Â | Â |
E. coli Bl21star(DE3) | B F- ompT hsdSB(rB-mB-) gal dcm rne131 (DE3) | Invitrogen |
E. coli Bl21(DE3)-RIL | B F- ompT hsdS(rB-mB-) dcm Tetr gal λ(DE3) endA Hte [argU ileY leuW Camr] | Stratagene |
Plasmid | Â | Â |
pCIP | Ampr, T7 promoter, lacO, ColE1 ori, parental DNA, replacing pgm gene with wt6pgdh, co6pgdh or g6pdh | [44] |
pET21a | Â | Epoch Biolabs |
pET-trx-wt6pgdh | Ampr, T7 promoter, lacO, ColE1 ori, Trx-wt6pgdh | This study |
pET-ci-wt6pgdh | wt6pgdh gene subcloned into pCIP | This study |
pET-ci-co6pgdh | co6pgdh gene subcloned into pCIP | This study |
pET-co6pgdh-his | co6pgdh gene with C-terminal (His)6 cloned into pET21a | Â |
pET-co6pgdh | Removed C-terminal (His)6 from pET-co6pgdh-his | This study |
pET-ci-g6pdhp | Expression of T. maritima g6pdh | This study |
pET26b-xr | Expression of N. crassa xylose reductase | [57] |
Primers* | Â | Final plasmid |
Trx-F | 5'-caccatggtgaaatctcatattggtctcatcggtc-3' | pET-trx-wt6pgdh |
Trx-R | 5'-tcatcctatctctccttcctcccagttg-3' | Â |
CI-wt-F | 5'-ccagtctactcgag gtgaaatctcatattggtctcatcggtc-3' | pET-ci-wt6pgdh |
CI-wt-R | 5'-ccagtctagtcgac cctatctctccttcctcccag-3' | Â |
CI-co-F | 5'-ccagtcta ctcgag ggctcttccatgaaatcccacattggcctgatc-3' | pET-ci-co6pgdh |
CI-co-R | 5'-ccagtctaggatcc tcaagtcgagccaatctccccctcctccc-3' | Â |
NH-F | 5'-gaggagggggagattggctaacatcaccaccaccattaag-3' | pET-co6pgdh |
NH-R | 5'-cttaatggtggtggtgatgttagccaatctccccctcctc-3' | Â |
G6P-F | 5'-ccagtctactcgag ggctcttcc atgaagtgcagtctgggattg-3' | pET-ci-g6pdh |
G6P-R | 5'-ccagtctagtcgac agttttctccattttctacc-3' | Â |