Difference between revisions of "Part:BBa K3515013:Design"
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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. | 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. | ||
| + | [[Image:spectragreencherry.png|650px]] | ||
| + | |||
| + | 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=== | ===Source=== | ||
Latest revision as of 17:21, 16 June 2020
Parathyroid Hormone Receptor with cysteine modification(s) and FRET to monitor PTH levels using a b
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 821
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 737
- 1000COMPATIBLE 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.
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.