Difference between revisions of "Part:BBa K4367011"
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<h2>Description</h2> | <h2>Description</h2> | ||
− | 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 | + | 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 dependent on the distance 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. <br><br> |
+ | FP consists of the two fluorescent proteins mNeongreen, and mRuby3. These proteins are fused together with a typical GS-linker, containing a cleavable sequence specific to TEVp, as shown in figure 1. <br><br> | ||
+ | |||
+ | <strong>Figure 1. Model of the initial system. mRuby3 is shown in right, and mNeongreen in left. TEVp is seen connecting the two proteins. </strong> | ||
[[File:FRET.png|600px]]<br><br> | [[File:FRET.png|600px]]<br><br> | ||
+ | |||
+ | If TEV is present, it will cleave the linker and separate the fluorescent protein pair. This will allow them to flow away from each other. The increased distance will lower the transfer of excitation energy, decreasing the FRET phenomena. Using a plate reader, this reduction in emission is able to be observed, giving a tangible measurement in TEVp activity. <br><br> | ||
+ | |||
+ | <h2>Usage</h2> | ||
− | |||
− | |||
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. | 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. |
Revision as of 18:11, 11 October 2022
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 dependent on the distance 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 fluorescent proteins mNeongreen, and mRuby3. These proteins are fused together with a typical GS-linker, containing a cleavable sequence specific to TEVp, as shown in figure 1.
Figure 1. Model of the initial system. mRuby3 is shown in right, and mNeongreen in left. TEVp is seen connecting the two proteins.
If TEV is present, it will cleave the linker and separate the fluorescent protein pair. This will allow them to flow away from each other. The increased distance will lower the transfer of excitation energy, decreasing the FRET phenomena. Using a plate reader, this reduction in emission is able to be observed, giving a tangible measurement in TEVp activity.
Usage
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