Composite

Part:BBa_K4275040:Design

Designed by: Zihuan Zhang   Group: iGEM22_GreatBay_SCIE   (2022-10-11)


pLAC4-KmarxMFα-TrEGIII-t-tTDH1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 331
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 331
    Illegal NheI site found at 89
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 331
    Illegal XhoI site found at 1252
    Illegal XhoI site found at 2830
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 331
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 331
    Illegal AgeI site found at 1603
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1475


Design Notes

1. The whole expression construct is assembled via goldengate assembly. The plasmid backbone, pLAC4 promoter, MFalpha, mTurquiose and tTDH1 terminator are firstly assembled as the vector for later assembly operations.

2. The CDS fragment is inserted into the previously assembled K.marxianus secretory expression vector via the enzymatic cleavage of the fragment termini by BsaI, and the subsequent creation of TTCT DNA overhang and the ATCC DNA overhang.

3. the pLAC4 promoter is an galactose-inducible promoter derived from K.marxianus whilst the tTDH1 terminator is derived from S.cerevisiae.

Source

Kluyveromyces marxianus DMKU3-1042
Trichoderma reesei ATCC 13631
Clostridium thermocellum (Dockerin-I domain)
Saccharomyces cerevisiae

References

1. Rajkumar, Arun S. et al. "Biological Parts For Kluyveromyces Marxianus Synthetic Biology". Frontiers In Bioengineering And Biotechnology, vol 7, 2019. Frontiers Media SA, https://doi.org/10.3389/fbioe.2019.00097.
2.“Mating Factor Alpha-1 [Kluyveromyces Marxianus] - Protein - NCBI.” National Center for Biotechnology Information, U.S. National Library of Medicine, https://www.ncbi.nlm.nih.gov/protein/QGN17207.1
3. Chang, Jui-Jen et al. "PGASO: A Synthetic Biology Tool For Engineering A Cellulolytic Yeast". Biotechnology For Biofuels, vol 5, no. 1, 2012. Springer Science And Business Media LLC, https://doi.org/10.1186/1754-6834-5-53.
4. "Part:Bba K2753052 - Parts.Igem.Org". Parts.Igem.Org, 2022, https://parts.igem.org/Part:BBa_K2753052.