Difference between revisions of "Part:BBa K3515008:Design"
Kody klupt (Talk | contribs) |
Kody klupt (Talk | contribs) |
||
| Line 8: | Line 8: | ||
===Design Notes=== | ===Design Notes=== | ||
Added a cysteine for immobilization (A26C) opposite to the active site region for linker arm immobilization. Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation. | Added a cysteine for immobilization (A26C) opposite to the active site region for linker arm immobilization. Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation. | ||
| + | |||
| + | 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). | ||
Latest revision as of 17:20, 25 May 2020
Potassium binding protein with cysteine modification(s) to bind to a biosensor and FRET to monitor p
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 766
Illegal AgeI site found at 823
Illegal AgeI site found at 883 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Added a cysteine for immobilization (A26C) opposite to the active site region for linker arm immobilization. Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation.
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 (U00096.3) along with our own modifications.
References
Shen, Y., Wu, S.Y., Rancic, V., Aggarwal, A., Qian, Y., Miyashita, S.I., Ballanyi, K., Campbell, R.E. and Dong, M., 2019. Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration. Communications biology, 2(1), pp.1-10.