Difference between revisions of "Part:BBa K3515011:Design"
Kody klupt (Talk | contribs) (→References) |
|||
| (One intermediate revision by the same user not shown) | |||
| Line 9: | Line 9: | ||
Added a Cysteine for immobilization (S224C) opposite to the active site region. Removed C34S, C120L, C169V, C201S, C235F, and C242T to ensure only one cysteine would bind to an immobilization linker arm. Amino acid substitution considerations were made using an intensive BLAST search to ensure conservation. | Added a Cysteine for immobilization (S224C) opposite to the active site region. Removed C34S, C120L, C169V, C201S, C235F, and C242T to ensure only one cysteine would bind to an immobilization linker arm. 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). | ||
===Source=== | ===Source=== | ||
| Line 16: | Line 20: | ||
===References=== | ===References=== | ||
| + | Choi, M., Yamada, S. and Makishima, M., 2011. Dynamic and ligand-selective interactions of vitamin D receptor with retinoid X receptor and cofactors in living cells. Molecular pharmacology, 80(6), pp.1147-1155. | ||
Latest revision as of 17:11, 29 May 2020
Vitamin D receptor binding protein with cysteine modification(s) to bind to a biosensor and FRET to
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 900
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Added a Cysteine for immobilization (S224C) opposite to the active site region. Removed C34S, C120L, C169V, C201S, C235F, and C242T to ensure only one cysteine would bind to an immobilization linker arm. 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 (J03258.1) along with our own modifications.
References
Choi, M., Yamada, S. and Makishima, M., 2011. Dynamic and ligand-selective interactions of vitamin D receptor with retinoid X receptor and cofactors in living cells. Molecular pharmacology, 80(6), pp.1147-1155.