Coding

Part:BBa_K3515007:Design

Designed by: Kody Klupt, Sono Khan   Group: iGEM20_Queens_Canada   (2020-05-25)


Troponin C, a calcium binding protein with cysteine modification(s) to bind to a biosensor and FRET


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1093
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Added a cysteine for immobilization (K93C) opposite to the active site region for linker arm immobilization and removed a cysteine previously existing (C101A). Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation. N109D, D111N, N145D, and D147N modifications were made based on previous evidence of these substitutions permitting selective calcium binding in the physiological range and removing the ability of magnesium to bind competitively.

An mNeonGreen and mCherry fluorophore pair was chosen due to a variety of reasons, including but not limited to; stability at varying pH, brightness, and a large dynamic linear range due to its intensity.

Spectragreencherry.png

Fluorophore spectrum of the mNeonGreen and mCherry fluorophores. Optical density data for wavelengths 300 nm to 750 nm were plotted for mNeonGreen and mCherry fluorescent proteins, in green and red colors, respectively. Data was obtained from www.fpbase.org. Excitation and emission peaks are labelled as EX and EM, respectively, for each fluorescent protein. Triangular dashed region shows the approximate fluorophore pair overlap, indicating that at an appropriate distance, energy transfer will occur between the donor (mNeonGreen) and acceptor (mCherry).


Source

The source of this part is its sequence retrieved from the European Nucleotide Archive (M19027.1) along with our own modifications.

References

Mank, M., Reiff, D.F., Heim, N., Friedrich, M.W., Borst, A. and Griesbeck, O., 2006. A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change. Biophysical journal, 90(5), pp.1790-1796