Part:BBa_K3515006:Design
Synechococcus Phosphate Binding Protein with cysteine modification(s) and FRET to monitor phosphate
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1539
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1129
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
- Added a Cysteine for immobilization (D256C) opposite to the active site region. Removed C135A and C189S 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. A T22A modification was made based on previous evidence of this substitution permitting phosphate binding in the physiological range. No modifications were made to the FRET acceptor or 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 (BX569691.1) along with our own modifications.
References
Gu, H., Lalonde, S., Okumoto, S., Looger, L.L., Scharff-Poulsen, A.M., Grossman, A.R., Kossmann, J., Jakobsen, I. and Frommer, W.B., 2006. A novel analytical method for in vivo phosphate tracking. FEBS letters, 580(25), pp.5885-5893.