Part:BBa_K3646007
enhancedCFP CDS
Coding sequence of enhanced Cyan Fluorescent Protein, mammalian codon optimised.
We will be using this part in concert with BBa_K3646002 as a FRET reporter in our project FRaPPe to study and quantify our PPI of choice. Our selected FRET pair is CFP-YFP. Cyan donors have high quantum yield. Yellow Fluorescent Proteins are photostable and less sensitive to pH changes.
FRET (Förster or Fluorescence Resonance Energy Transfer) is a technique to assess a wide range of biological activities such as protein-protein interactions, conformational changes, enzymatic processes and so on, all of which involve molecular proximity. Essentially functioning as a spectroscopic ruler, this phenomenon employs two fluorophores (donor and acceptor) wherein the excitation energy of the donor is transmitted to the acceptor in its vicinity, resulting in the distinctive fluorescence emission spectra of the acceptor. These biosensors are highly sensitive to the separation distance between the interacting components (within 1 to 10 nm range), and this in turn dictates many of the factors to be considered while finding appropriate FRET pairs for the biosensor of interest. An advantage that FRET confers over other reporter assays in that it allows observation of live cells in a non-destructive and minimally invasive way.
FRET is most appropriately quantified using the FRET efficiency, which serves as an instrument-independent index. In living cells expressing proteins fused with FPs, FRET efficiency can be accurately determined using donor fluorescence lifetime or sensitized acceptor emission intensity. However, equating FRET efficiency with protein-protein interaction presents a challenge, as FRET efficiency is a function of multiple variables.
Hence, the measured FRET efficiency per se is not an appropriate estimator of the protein-protein binding affinity. Estimation of the binding affinity is an important goal of protein-protein interaction assays. The relative Kd of two proteins tagged with genetically encoded fluorophores expressed in living cells is thus required, and there are methods to compute this from the experimental FRET measurements. (Chen at al., 2007)
That being said, there have been studies to determine if the apparent FRET efficiency can reliably and rapidly estimate K D for a set of structurally diverse PPIs without the need for extensive data fitting (Lin et al., 2018). It has been found that within the affinity range of mM–nM, a near linear relationship between the FRET efficiency (E) and the affinity (pKd) exists.
In our project, the process we will follow involves titrating our engineered cell culture with the peptide inhibitor and measuring the FRET emission. We will get an emission vs log(concentration) curve from which we will obtain IC50 values [ just the concentration of the inhibitor at which the emission becomes 50 % of the maximum ].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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