Part:BBa_K4814006
ATRIP-EGFP
FRET is using fluorescent proteins as probes to detect the interaction of targeted proteins. The distance-dependent process transfers energy from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) through intermolecular long-range dipole–dipole coupling once the desired proteins bind (Sekar, R. B. and Periasamy, A., 2003). The critical Förster radius (typically 3-6 nm) at angstrom distances (10–100 Å) can be calculated to increase the accuracy and ensure precise energy transfer. (Alan Mulllan, n.d.) By using FRET, we can therefore observe the interaction of two proteins by measuring the lifetime of the fluorescent proteins attached to them.
As the aim of this design is to detect DNA damages in mammalian cells, we have used CMV promoter and the Lenti virus vector. Please refer to BBa_K4814004 and BBa_K4814005 (ATRIP and RPA1) for detailed explanation of the two proteins involved in the DNA damage checkpoint process.
The EGFP is derived from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC146266/ (same as BBa_K1875003), a mammalian codon optimized enhanced GFP.
After exposing the cells to a UVB dosage of 100 J/m^2, we observed aggregation of the EGFP signal (Fig. 1 and 2). Interestingly, fluorescence was detected in both the Green and Red channels. It is important to note that the emission of GFP is dependent on its fluorescence spectra, as mentioned in studies by Sattarzadeh, A. et al. (2015) and Licea-Rodriguez, J. (2019). This fluorescence could potentially be attributed to GFP emitting at around 560 nm.
|
|
| None |