Part:BBa_K4244046

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.

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]