Difference between revisions of "Part:BBa K1319002"

Revision as of 01:09, 17 October 2014

RFC[25]-compatible dark quencher based on K1319000 (E0030)

This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP) called REACh 2.

Three mutations were introduced that eliminated fluorescence:

• L90I
• Y145W
• H148R

References

• Ganesan S, Ameer-Beg SM, Ng TT, Vojnovic B, Wouters FS. A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Förster resonance energy transfer with GFP. Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4089-94. Epub 2006 Mar 6. PubMed PMID: 16537489; PubMed Central PMCID: PMC1449651. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1449651/?report=classic PubMed Central]

Usage and Biology

This protein is designed to be a dark quencher for GFP (E0040) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick K1319014 this is realised and the proteins are fused with the linker K1319016 which includes a specific TEV protease (available as K1319004) cleavage site. The fusion of the proteins brings GFP and REACh 2 in proximity to each other which allows GFP and REACh 2 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh 2 and released as thermal energy instead of visible light. This eliminates the GFP fluorescence and allows for a release of a strong fluorescence signal if a TEV protease is expressed and the linker is cut. The cutting separates GFP and REACh 2 cancelling the FRET interaction and providing a GFP fluorescence response.

Sequence and Features