Coding

Part:BBa_K4275013:Design

Designed by: Kejia Miao   Group: iGEM22_GreatBay_SCIE   (2022-09-29)


OlpB-3C-Ag3


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 706
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1252
    Illegal NgoMIV site found at 1341
    Illegal AgeI site found at 183
    Illegal AgeI site found at 319
    Illegal AgeI site found at 370
    Illegal AgeI site found at 502
    Illegal AgeI site found at 868
    Illegal AgeI site found at 886
    Illegal AgeI site found at 1312
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1069
    Illegal SapI.rc site found at 1081
    Illegal SapI.rc site found at 1705


Design Notes

1. A 6x His affinity purification tag(HHHHHH) 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 are interspaced with a 10 aa flexible GS linker GGGGSGGGGS to avoid the steric inhibtion of the giant OlpB scaffold.

5. DNA sequence is codon-optimized based on the codon-usage table of E.coli Strain K12.MG1655.


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.