Coding

Part:BBa_K3515013:Design

Designed by: Kody Klupt, Sono Khan   Group: iGEM20_Queens_Canada   (2020-06-16)


Parathyroid Hormone Receptor with cysteine modification(s) and FRET to monitor PTH levels using a b


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 821
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 737
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Isolated the N- terminal extracellular domain of PTHR, residues 1-60 for binding to PTH. Kept the one C48 residue in place to bind a corresponding cysteine linker arm (for biosensor immobilization). An intensive BLAST search was done to ensure that the C48 residue did not form a crucial interaction with the ligand. No modifications were made to the FRET donor.

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 (V00303.1) along with our own modifications.

References

Nie, Y., Li, J., Liu, Y., Zhang, Q. and Ma, Q., 2020. A Visual FRET Immunofluorescent Biosensor for Ratiometric Parathyroid Hormone (1–84) Antigen Point-of-Care Detection. Journal of Fluorescence, pp.1-6.