Coding

Part:BBa_K4275005:Design

Designed by: Wang Sze Ting   Group: iGEM22_GreatBay_SCIE   (2022-09-29)


MtCDH-t


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2706
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1507
    Illegal AgeI site found at 358
    Illegal AgeI site found at 745
    Illegal AgeI site found at 871
    Illegal AgeI site found at 1420
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

1. The catalytic domain and the dockerin domain is interspaced with a glycine-rich linker.

2. The 79 amino acid long type-I dockerin domain is fused at the terminal of the GS linker, followed by a TEV site and a 8xhis affinity purification tag (HHHHHHHH).

3. DNA sequence is codon-optimized based on the codon-usage table of E.coli Strain K12.MG1655, but the protein was later expressed in K. marxianus due to the lack of post-translational modifications and the formation of inclusion bodies in E.coli BL21(DE3).

4. The cytochrome domain of this enzymatic construct (Figure 1) facilitates the oxidation of cellobiose and the electron transfer from cellobiose to the copper ion of LPMO (Lyctic Polysaccharide Monooxygenase) BBa_K4275003.

Source

Myceliophthora thermophila ATCC 42464
Clostridium thermocellum (Dockerin-I domain)

References

1. Phillips, Christopher M. et al. "Cellobiose Dehydrogenase And A Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation By Neurospora Crassa". ACS Chemical Biology, vol 6, no. 12, 2011, pp. 1399-1406. American Chemical Society (ACS), https://doi.org/10.1021/cb200351y.