Difference between revisions of "Part:BBa K1319001"

Revision as of 21:29, 16 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)..

Two mutations were introduced that eliminated fluorescence:

L90I
Y145W

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 K1319013 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 bring GFP and REACh 1 in proximity to each other which allows GFP and REACh 1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh 1 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 1 cancelling the FRET interaction and providing a GFP fluorescence response.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+This protein is designed to be a dark quencher for GFP ([https://parts.igem.org/Part:BBa_E0040 E0040]) in a FRET system. When used in a fusion protein with GFP it reduces the observed fluorescence of GFP drastically. In the biobrick [https://parts.igem.org/Part:BBa_K1319013 K1319013] this is realised and the proteins are fused with the linker [https://parts.igem.org/Part:BBa_K1319016 K1319016] which includes a specific TEV protease (available as [https://parts.igem.org/Part:BBa_K1319004 K1319004]) cleavage site. The fusion of the proteins bring GFP and REACh 1 in proximity to each other which allows GFP and REACh 1 to act as donors and acceptors in a FRET (Förster Energy Transfer System) system. GFPs emission energy is thereby taken up by REACh 1 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 1 cancelling the FRET interaction and providing a GFP fluorescence response.
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