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Table 1 The strains, plasmids, and oligonucleotides used in this study

From: Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration

Description

Contents

Reference/sources

Strain

  

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

  

pCIP

Ampr, T7 promoter, lacO, ColE1 ori, parental DNA,

replacing pgm gene with wt6pgdh, co6pgdh or g6pdh

[44]

pET21a

 

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

 

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*

 

Final plasmid

Trx-F

5'-caccatggtgaaatctcatattggtctcatcggtc-3'

pET-trx-wt6pgdh

Trx-R

5'-tcatcctatctctccttcctcccagttg-3'

 

CI-wt-F

5'-ccagtctactcgag gtgaaatctcatattggtctcatcggtc-3'

pET-ci-wt6pgdh

CI-wt-R

5'-ccagtctagtcgac cctatctctccttcctcccag-3'

 

CI-co-F

5'-ccagtcta ctcgag ggctcttccatgaaatcccacattggcctgatc-3'

pET-ci-co6pgdh

CI-co-R

5'-ccagtctaggatcc tcaagtcgagccaatctccccctcctccc-3'

 

NH-F

5'-gaggagggggagattggctaacatcaccaccaccattaag-3'

pET-co6pgdh

NH-R

5'-cttaatggtggtggtgatgttagccaatctccccctcctc-3'

 

G6P-F

5'-ccagtctactcgag ggctcttcc atgaagtgcagtctgggattg-3'

pET-ci-g6pdh

G6P-R

5'-ccagtctagtcgac agttttctccattttctacc-3'

 
  1. * Underlined nucleotide sequences indicate restriction endonuclease sites.