Difference between revisions of "Part:BBa K4367011"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4367011 short</partinfo>
 
<partinfo>BBa_K4367011 short</partinfo>
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WORK IN PROGRESS:
 
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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).
 
FRET protein (FP) is a reporter system which allows for the measurement of the catalytic activity of the Tobacco Etch Virus Protease (TEVp).
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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>
 
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>
  
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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>
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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.  
  
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<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>
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<strong>Figure 1. Model of the initial system. mRuby3 is shown in right, and mNeongreen in left. TEVp is seen connecting the two proteins. The model has been produced using AlphaFold. </strong> <br><br>
 
[[File:FRET.png|600px]]<br><br>
 
[[File:FRET.png|600px]]<br><br>
  
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<h2>Usage</h2>
 
<h2>Usage</h2>
  
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With the addition of TEVp to the FP system, the linker connecting mRuby3 and mNeongreen will be severed. This will separate the proteins from each other, as shown in figure 2 and 3. <br><br>
  
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<strong>Figure 2. Model of mRuby3 with attached linker cleaved by TEVp. </strong> <br><br>
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[[File:mRuby3.png|600px]]<br><br>
  
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<strong>Figure 3. Model of mNeongreen with attached linker cleaved by TEVp. </strong> <br><br>
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[[File:mNeongreen.png|350px]]<br><br>
  
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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.
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With the two proteins now independent, the FRET phenomena will decrease, which can be measured using a plate reader. When measuring the fluorescence, the maximum emission and excitation wavelengths should be used. These are seen in table 1. <br><br>
 +
 
 +
<strong>Table 1. The maximum excitation and emission wavelengths of mRuby3 and mNeongreen [2]. </strong> <br><br>
 +
[[File:table1.png|600px]]<br><br>
 +
 
 +
For FP, the donor is mNeongreen, and the acceptor is mRuby3. As such, measurement should be done at 592 nm, with light emitted at 506 nm. The ability to now measure TEVp activity, allows for further investigation into the whole Cell Free system. The activity of the combined TEVp halves can be measured, and compared to that of regular non-split TEVp. Results of a working recombinant TEVp can further test the function of the inhibited Beta-galactosidase (iGal) (https://parts.igem.org/Part:BBa_K4367009) for the Cell Free system, by ruling out TEVp as the source of error. <br><br>
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 +
<h2>Future Design Considerations</h2>
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 +
The excitation and emission of each fluorescent protein must be appropriate to induce FRET. The proteins mRuby3 and mNeongreen were chosen, as studies have shown them to be stable, and give clear signals [2]. This choice was further validated using the website FPbase [3] which allowed for the appropriate excitation and emission wavelengths to be found.
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There are however many other fluorescent proteins which can be used, some which might give clearer and more stable observations. For example, mTurqoise2 has been reported to have a higher quantum yield than mNeongreen; a key variable to determining FRET efficiency [1].
 +
 
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K4367011 SequenceAndFeatures</partinfo>
  
  
 
<h2>References</h2>
 
<h2>References</h2>
  
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[1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038762/#B3-sensors-16-01488
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[1] Bajar, B., Wang, E., Zhang, S., Lin, M. and Chu, J., 2016. A Guide to Fluorescent Protein FRET Pairs. Sensors, [online] 16(9). Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038762/#B3-sensors-16-01488> [Accessed 11 October 2022]. <br><br>
 +
 
 +
[2] Bajar, B., Wang, E., Lam, A., Kim, B., Jacobs, C., Howe, E., Davidson, M., Lin, M. and Chu, J., 2016. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Scientific Reports, [online] 6. Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754705/> [Accessed 11 October 2022]. <br><br>
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[3] FPbase. n.d. FPbase FRET Calculator. [online] Available at: <https://www.fpbase.org/fret/> [Accessed 11 October 2022].
  
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4367011 SequenceAndFeatures</partinfo>
 
  
  

Revision as of 19:28, 11 October 2022


FRET Protein (FP)

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. The model has been produced using AlphaFold.

FRET.png

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

With the addition of TEVp to the FP system, the linker connecting mRuby3 and mNeongreen will be severed. This will separate the proteins from each other, as shown in figure 2 and 3.

Figure 2. Model of mRuby3 with attached linker cleaved by TEVp.

MRuby3.png

Figure 3. Model of mNeongreen with attached linker cleaved by TEVp.

MNeongreen.png

With the two proteins now independent, the FRET phenomena will decrease, which can be measured using a plate reader. When measuring the fluorescence, the maximum emission and excitation wavelengths should be used. These are seen in table 1.

Table 1. The maximum excitation and emission wavelengths of mRuby3 and mNeongreen [2].

Table1.png

For FP, the donor is mNeongreen, and the acceptor is mRuby3. As such, measurement should be done at 592 nm, with light emitted at 506 nm. The ability to now measure TEVp activity, allows for further investigation into the whole Cell Free system. The activity of the combined TEVp halves can be measured, and compared to that of regular non-split TEVp. Results of a working recombinant TEVp can further test the function of the inhibited Beta-galactosidase (iGal) (https://parts.igem.org/Part:BBa_K4367009) for the Cell Free system, by ruling out TEVp as the source of error.

Future Design Considerations

The excitation and emission of each fluorescent protein must be appropriate to induce FRET. The proteins mRuby3 and mNeongreen were chosen, as studies have shown them to be stable, and give clear signals [2]. This choice was further validated using the website FPbase [3] which allowed for the appropriate excitation and emission wavelengths to be found.

There are however many other fluorescent proteins which can be used, some which might give clearer and more stable observations. For example, mTurqoise2 has been reported to have a higher quantum yield than mNeongreen; a key variable to determining FRET efficiency [1].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1504
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 14
    Illegal BsaI.rc site found at 1511


References

[1] Bajar, B., Wang, E., Zhang, S., Lin, M. and Chu, J., 2016. A Guide to Fluorescent Protein FRET Pairs. Sensors, [online] 16(9). Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038762/#B3-sensors-16-01488> [Accessed 11 October 2022].

[2] Bajar, B., Wang, E., Lam, A., Kim, B., Jacobs, C., Howe, E., Davidson, M., Lin, M. and Chu, J., 2016. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Scientific Reports, [online] 6. Available at: <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754705/> [Accessed 11 October 2022].

[3] FPbase. n.d. FPbase FRET Calculator. [online] Available at: <https://www.fpbase.org/fret/> [Accessed 11 October 2022].