Part:BBa_K4244048

flip-spisPink

This flip-spisPink 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 spisPink structure. MMP-9 recognizes the cleavage site and cuts off the K5 domain, restoring spisPink 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 flip-spisPink was predicted using alphafold as shown below.

The left shows uncleaved flip-spisPink, the middle the cleaved, active flip-spisPink, and the right regular spisPink

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 655
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]