Composite

Part:BBa_K4275042:Design

Designed by: Wei Zijian   Group: iGEM22_GreatBay_SCIE   (2022-10-12)


pT7-RBS-OlpB-Ag3-tT7


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2058
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 746
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1292
    Illegal NgoMIV site found at 1381
    Illegal AgeI site found at 223
    Illegal AgeI site found at 359
    Illegal AgeI site found at 410
    Illegal AgeI site found at 542
    Illegal AgeI site found at 908
    Illegal AgeI site found at 926
    Illegal AgeI site found at 1352
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1109
    Illegal SapI.rc site found at 1121
    Illegal SapI.rc site found at 1745


Design Notes

1. A 6x His affinity purification tag(HHHHHH) is added to N-terminal following the start codon, to allow for Ni-NTA purification.

2. The number of cohesins is cut from 7 to 3 to reduce cellular pressure, ensuring successful heterologous expression in E.coli

3. The original anchoring domain of OlpB that binds to yeast surface, glycosylphosphatidylinositol (ScGPI), is replaced by Ag3 so that OlpB can attach to the cell surface of E.coli through the interaction between Ag3 and Nb3 cell surface expression system BBa_K4275026.

4. The OlpB scaffoldin domain and the Ag3 antigen domain iare interspaced with a 10 aa flexible GS linker GGGGSGGGGS.


Source

Clostridium thermocellum

References

1. Anandharaj, Marimuthu et al. "Constructing A Yeast To Express The Largest Cellulosome Complex On The Cell Surface". Proceedings Of The National Academy Of Sciences, vol 117, no. 5, 2020, pp. 2385-2394. Proceedings Of The National Academy Of Sciences, https://doi.org/10.1073/pnas.1916529117.