Coding

Part:BBa_K4244046

Designed by: Max Allewijn   Group: iGEM22_Wageningen_UR   (2022-10-08)
Revision as of 12:39, 12 October 2022 by Sarah99 (Talk | contribs)

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flipUM (MMP-9 inducible)

This flipUM is designed in such a way that it will only regain its colour when it is cleaved by MMP-9. Two inserts have been made, the E5 and K5 domain. These two domains dimerize and disrupt GFP structure. MMP-9 recognizes the cleavage site and cuts off the K5 domain, restoring Ultramarine function.

The flip design is based off the following paper: Zhang, Q., Schepis, A., Huang, H., Yang, J., Ma, W., Torra, J., Zhang, S. Q., Yang, L., Wu, H., Nonell, S., Dong, Z., Kornberg, T. B., Coughlin, S. R., & Shu, X. (2019). Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals. Journal of the American Chemical Society, 141(11), 4526–4530. https://doi.org/10.1021/JACS.8B13042/SUPPL_FILE/JA8B13042_SI_002.PDF

The structure of flipUM was predicted using alphafold as shown below.

Flip ultramarine.png

The left shows uncleaved flipUM, the middle the cleaved, active flipUM, and the right regular UM


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 423
    Illegal EcoRI site found at 514
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 423
    Illegal EcoRI site found at 514
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 423
    Illegal EcoRI site found at 514
    Illegal BglII site found at 625
    Illegal XhoI site found at 682
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 423
    Illegal EcoRI site found at 514
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 423
    Illegal EcoRI site found at 514
    Illegal AgeI site found at 543
    Illegal AgeI site found at 789
  • 1000
    COMPATIBLE WITH RFC[1000]


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