Composite

Part:BBa_K4759271

Designed by: Jianghua Chen   Group: iGEM23_Jiangnan-China   (2023-10-08)
Revision as of 17:38, 11 October 2023 by JianghuaChen (Talk | contribs) (→‎Usage and Biology)


T7-RBS6-pntAB-RBS2-nadK

Design Notes

We cloned nadK and pntAB genes to construct the plasmid pACYCDuet-pntAB-nadK.

Usage and Biology

P450 enzyme catalysis required redox partners to transfer NADPH electrons to heme to catalyze the substrate. The content of NADPH in E. coli cells was low, which can only meet its own growth needs. By constructing a cofactor circulatory system, the effect of NADPH on whole-cell activity was investigated. However, the cost of exogenous NADPH addition is higher and the catalytic efficiency is low. NAD+ kinase (NadK) and membrane-bound hydrogenase (PntAB) can be used to enhance the circulation of NADPH. Thus, 3 recombinant strains are constructed. The recombinant plasmid pACYCDuet-nadK is expressed in E. coli C1 (C2 strain). The recombinant plasmid pACYCDuet-pntAB (BBa_K4759269) was expressed in E. coli C1 (C3 strain). The recombinant plasmid pACYCDuet-pntAB-nadK(BBa_K4759271) was expressed in E. coli C1 (C4 strain). Without the addition of NADPH, the conversion rate of whole-cell catalysts prepared by the C4 strain was as high as 99.1%. Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
    Illegal EcoRI site found at 1626
    Illegal PstI site found at 2233
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
    Illegal EcoRI site found at 1626
    Illegal PstI site found at 2233
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
    Illegal EcoRI site found at 1626
    Illegal BglII site found at 2158
    Illegal BamHI site found at 1620
    Illegal XhoI site found at 720
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
    Illegal EcoRI site found at 1626
    Illegal PstI site found at 2233
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 714
    Illegal EcoRI site found at 1353
    Illegal EcoRI site found at 1626
    Illegal PstI site found at 2233
    Illegal NgoMIV site found at 1190
  • 1000
    COMPATIBLE WITH RFC[1000]


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