Coding

Part:BBa_K3147011:Design

Designed by: Thomas Bessede   Group: iGEM19_Montpellier   (2019-10-14)

Mutant TEV (PH21) protease fused to anti-sfGFP VHH with TEV-cleavable maltose binding protein


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 77
    Illegal BsaI.rc site found at 2377
    Illegal SapI.rc site found at 1868


Design note

The TEV protease used is a mutant TEV with the following mutation: T17A, T146A, D148P, S153C, S168T, S170A, T173A, S219P. [3]

The TEV cleavage site used is ENLYFH/G with / indicating the peptide cleavage. [3]

The Terminator used is a double terminator (Part:BBa_B0015).

The VHH is a VHH directed against the sfGFP, it recognizes a conformational pattern. [4]

Reference

[3] Yi, L. et al. 2013. « Engineering of TEV Protease Variants by Yeast ER Sequestration Screening (YESS) of Combinatorial Libraries ». Proceedings of the National Academy of Sciences 110(18): 7229‑34

[4] Kubala, M. H., Kovtun, O., Alexandrov, K., & Collins, B. M. (2010). Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein science : a publication of the Protein Society, 19(12), 2389–2401. doi:10.1002/pro.519