Part:BBa_K4367011
FRET Protein (FP)
WORK IN PROGRESS: use pictures of FP from here: https://docs.google.com/document/d/1C8z9Y3Bf5k2NkXp4JD_NCtEUfyfqcI9ZZiLw7pQM-yQ/edit
FRET protein (FP) is a reporter system which allows for the measurement of the catalytic activity of the Tobacco Etch Virus Protease (TEVp).
Description
Fluorescence Resonance Energy Transfer (FRET) is a technique utilizing fluorescent protein pairs, working as a biosensor for changes in molecular proximity [1]. When in an excited state, one of the proteins (the donor) will transfer this excitation energy to the other protein (the acceptor), causing it to fluoresce its specific spectrum. FRET systems are very dependant on the distace between donor and acceptor, being sensitive to ranges of 1-10 nm [1]. This sensitivity allows for the reporting of conformational changes, and enzymatic activity.
FP consists of the two flourescent proteins mNeongreen, and mRuby3. These proteins are fused together with a typical GS-linker, containing a cleavable sequence specific to TEVp.
that are fused together with a cleavable linker. The linker is a typical GS-linker and Tobacco Etch Virus Protease is able to cleave a target sequence within the linker. If TEV is present, it will cleave the linker and separate pair of fluorescent proteins, which will decrease the FRET phenomena. With a plate reader it will be possible to observe this.
FP acts as a reporter protein that measures the catalytic activiy of a protease. It would be relativly easy to quantify if FP is expressed as it is fluorescent, which can be used to troubleshoot if the protease is functional or not with relative ease. For example, it would be possible to check if TEVp works, and if it does, it would give information if iGal works when it is mixed with TEVp, and so on for following proteins.
References
[1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038762/#B3-sensors-16-01488
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1504
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 14
Illegal BsaI.rc site found at 1511
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