Difference between revisions of "Part:BBa K4814008"

Revision as of 05:02, 10 October 2023

ATRIP-ECFP

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 Lentivirus 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. ATRIP-CFP is co-infected with RPA1-YFP to conduct FRET.

We transfected with ATRIP-ECFP and ATRIP-ECFP + RPA1-YFP (C+Y). However, due to time constraints images of the cells were taken in the six-well plate and the quality was slightly compromised. Nonetheless, these images still provide confirmation that both vectors were successfully transfected into the cells. The CFP fluorescence was excited at 405 nm and the YFP fluorescence was excited at 488 nm.

Figure 3. The images of ATRIP-ECFP and ATRIP-ECFP + RPA1-EYFP (C+Y) on six-well plate.

Sekar, R. B., & Periasamy, A. (2003). Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. The Journal of cell biology, 160(5), 629–633. https://doi.org/10.1083/jcb.200210140

Alan Mulllan. (n.d.). Advanced microscopy applications – an overview of FRET. OXFORD instruments. https://andor.oxinst.com/learning/view/article/fret

Yang, T. T., Cheng, L., & Kain, S. R. (1996). Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic acids research, 24(22), 4592–4593. https://doi.org/10.1093/nar/24.22.4592

Sequence and Features