Difference between revisions of "Team:NYMU-Taipei/Model"

 
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<div class="column">
 
<div class="column">
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<h1 style="color:#638695; font-size:70px; line-height:1em;"> Interviews and Visits </h1>
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<div class="story section">
 
<div class="story section">
−
<h2 id="1">2018/1/26 </h2>
+
<h1 id="1" class="story-title subtitle">FRET Ratio Model</h1>
−
<h2 class="story-title subtitle">FIRDI</h2>
+
<img src="https://static.igem.org/mediawiki/2018/6/6d/T--NYMU-Taipei--model-1.png" style="width:500px;">
−
<img src="https://static.igem.org/mediawiki/2018/b/ba/T--NYMU-Taipei--hp-FIRDI.jpg" style="box-shadow: -20px -20px  #4B727B;">
+
<img src="https://static.igem.org/mediawiki/2018/1/1e/T--NYMU-Taipei--FRETRatio1.png" style="width:300px;">
 
<div class="paragraphs">
 
<div class="paragraphs">
−
<p >In the early morning of Jan 26th, we hurried from Taipei to Hsinchu to visit Food Industry Research and Development Institute(FIRDI). Under the guidance of senior researchers in the institute, we came to know the importance of microorganisms in food industry and the institute’s dedication to preserving biological resources in the past 50 years.</p>
+
<h3>Background</h3>
−
<p>More importantly, we discussed with three experts, research scientist Guey-Yuh Liou, senior research scientist Hing-Yuen Chan, and associated research scientist Li-Wen Yu, ideas and topics we generated last year in hopes of diminishing the obstacles we were faced with. The experts thought through the ideas with us and suggested possible problems and potential market demands of the topics. With the help of the experts, we acquired numerous inspirations and new directions. </p>
+
<p >This model aims to find out how much FRET protein should be added into our screening system.</p>
−
<p>We are thankful for the help from the experts in FIRDI and Dr. Chou Tzu-Ho, who led this visit to the institute. What we have learned  will continue to foster our ideas and projects.</p>
+
<h3>Methods</h3>
 +
<p >Chemical equilibrium is used to determine the concentration of each protein, the fluorescence level, and the minimal FRET protein activity required to produce the fluorescence that can be detected. This model assumes the portion of active protein in all proteins is constant. The equilibrium constants are from Victoria E. Ahn's study [1] and Zhihong Cheng's study [2]. The limited experiment budget for making FRET proteins is considered in this model.</p>
 +
<h3>Results</h3>
 +
<p> The relationship between fluorescence and DKK1 expression level </p>
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/a/af/T--NYMU-Taipei--FRETRatioResult_1.mov" autoplay="true" muted></video>
 +
<p> The relationship between fluorescence and the activity of one of the FRET proteins
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/4/4d/T--NYMU-Taipei--FRETRatioResult2.png" autoplay="true" muted></video>
 +
<p> The plot of the optimal activity of a FRET protein vs. the other </p>
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/8/8a/T--NYMU-Taipei--FRETRatioResult3.mov" autoplay="true" muted></video>
 +
<p> 3D plot of the fluorescence, the objective function we want to maximize. </p>
 +
<p> The x-axis is the concentration of one of the FRET protein and y-axis is that of the other FRET protein. </p>
 +
<img src="https://static.igem.org/mediawiki/2018/f/f5/T--NYMU-Taipei--FRETRatioResult4.png">
 +
<p> Contour a plot of the fluorescence, the objective function we want to maximize. </p>
 +
<p> The x-axis is the concentration of one of the FRET protein and y-axis is that of the other FRET protein. </p>
 +
<img src="https://static.igem.org/mediawiki/2018/9/9a/T--NYMU-Taipei--FRETRatioResult5.png">
 +
<p> A plot of the optimal “cheaper FRET protein” activity, vs. budget </p>
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/e/e4/T--NYMU-Taipei--FRETRatioResult9.mov" autoplay="true" muted></video>
 +
<p>A plot of the optimal “more expensive FRET protein” activity, vs. budget </p>
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/7/70/T--NYMU-Taipei--FRETRatioResult6.mov" autoplay="true" muted></video>
 +
<p> A plot of the optimal ratio vs. budget </p>
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/8/89/T--NYMU-Taipei--FRETRatioResult7.png" autoplay="true" muted></video>
 +
<br>
 +
K<sub>d</sub> values obtained from the references:
 +
<img src="https://static.igem.org/mediawiki/2018/1/19/T--NYMU-Taipei--FRETRatioResult8.png">
 +
 
 +
<h3>Conclusion</h3>
 +
<p >The optimal ratio of the amount of one FRET protein to that of the other is as the following:</p>
 +
<img src="https://static.igem.org/mediawiki/2018/c/cd/T--NYMU-Taipei--12.PNG">
 +
<p >The figures in the table indicate the optimal ratio of the protein on the top over the protein on the left.</p>
 
<p class="detailtrigger" id="1t" onclick="detail(event)">Click Here For More Info</p>
 
<p class="detailtrigger" id="1t" onclick="detail(event)">Click Here For More Info</p>
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</div>
 
 
</div>
 
</div>
 
<div class="detail story" id="1d">
 
<div class="detail story" id="1d">
 +
<embed src="https://static.igem.org/mediawiki/2018/a/a5/T--NYMU-Taipei--model-fret-ratio.pdf" style="width:800px; height:1200px; text-align:center;">
 +
</div>
 +
</div>
 +
 +
 +
<div class="story section">
 +
<h1 id="2" class="story-title subtitle">FRET Efficiency Model</h1>
 +
<img src="https://static.igem.org/mediawiki/2018/7/7a/T--NYMU-Taipei--FRETEfficiency1.png">
 +
 +
<div class="paragraphs">
 +
 +
<h3>Background</h3>
 +
<p >The efficiency of FRET is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Therefore, simulating structure of protein-protein interactions is important. This model aims to predict the FRET efficiency in order to determine which molecules and which termini should be used.</p>
 +
<h3>Methods</h3>
 +
<p >pyDockWEB [3] is used for structural prediction of protein-protein interactions and the prediction of distances between donor and acceptor. Förster theory is used to determine the FRET efficiency. YPet and CyPet are added to either N-terminus or C-terminus of each of the 2 FRET proteins.</p>
 +
<h3>Results</h3>
 +
<p> The protein-protein interactions structural predictions are as follows: </p>
 +
<img src="https://static.igem.org/mediawiki/2018/7/7a/T--NYMU-Taipei--FRETEfficiency1.png">
 +
<p> Cyan: LRP6-BP1BP2  &nbsp;&nbsp;&nbsp;&nbsp; Green: LRP6-BP3BP4 &nbsp;&nbsp;&nbsp;&nbsp;  Purple: DKK1 </p>
 
<br>
 
<br>
 +
<img src="https://static.igem.org/mediawiki/2018/9/98/T--NYMU-Taipei--FRETEfficiency2.png">
 +
<p> Cyan: LRP6-BP3BP4  &nbsp;&nbsp;&nbsp;&nbsp;  Green: VHH G5 &nbsp;&nbsp;&nbsp;&nbsp;  Purple: DKK1 </p>
 
<br>
 
<br>
−
<div class="paragraphs">aaaaaaaa</div>
+
<img src="https://static.igem.org/mediawiki/2018/3/33/T--NYMU-Taipei--FRETEfficiency3.png">
 +
<p> Cyan: VHH H7  &nbsp;&nbsp;&nbsp;&nbsp;  Green: VHH G5 &nbsp;&nbsp;&nbsp;&nbsp;  Purple: DKK1 </p>
 +
<p> The predicted distances are </p>
 +
<img src="https://brucechen034020.github.io/IHSCA/igem_model_14.png" style="width:800px;">
 +
<p> YPet and CyPet are added to the N-terminus or the C-terminus. </p>
 
<br>
 
<br>
 +
<h3>Conclusion</h3>
 +
<p >The FRET efficiency is given by the following table.</p>
 +
<img src="https://brucechen034020.github.io/IHSCA/igem_model_15.png" style="width:800px; float:left;">
 +
<p> YPet and CyPet are added to the N-terminus or the C-terminus. </p>
 
<br>
 
<br>
 +
 +
<p class="detailtrigger" id="2t" onclick="detail(event)">Click Here For More Info</p>
 +
</div>
 +
<div class="detail story" id="2d">
 +
The protein-protein interactions structural predictions can be downloaded from the following files <br>
 +
<a href="https://brucechen034020.github.io/IHSCA/DKK1-E3E4G5.pse"> DKK1-E3E4G5.pse </a>
 
<br>
 
<br>
 +
<a href="https://brucechen034020.github.io/IHSCA/DKK1E1E2E3E4.pse"> DKK1E1E2E3E4.pse </a>
 
<br>
 
<br>
 +
<a href="https://brucechen034020.github.io/IHSCA/DKK1E3E4.pdb"> DKK1E3E4.pdb </a>
 +
<br>
 +
<a href="https://brucechen034020.github.io/IHSCA/DKK1E3E4H7.pdb"> DKK1E3E4H7.pdb </a>
 +
<br>
 +
<a href="https://brucechen034020.github.io/IHSCA/DKK1G5H7-model-1.pse"> DKK1G5H7-model-1.pse </a>
 +
</div>
 +
</div>
 +
 +
 +
<div class="story section">
 +
<h1 id="3" class="story-title subtitle">mCherry Expression Model</h1>
 +
<br>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2018/f/f6/T--NYMU-Taipei--Reference1.png" style="width:400px; float:left;">
 +
 +
<img src="https://static.igem.org/mediawiki/2018/5/50/T--NYMU-Taipei--mCherry.jpg" style="width:400px; float:right;">
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
 +
<div class="paragraphs">
 +
<br>
 +
<br>
 +
<h3>Background</h3>
 +
<p >This project constructs a plasmid that connects DKK1 promoter to mCherry. The gene expression rate of DKK1 promoter is affected by testosterone activity and affects the expression level of mCherry. The expression level of mCherry should be greater than a threshold so that its fluorescence can be detected by devices. In order to achieve this threshold, sufficient amount of testosterone should be added into our screening system; this is the sensitivity of our screening system. This model aims to find out the sensitivity of our screening system.</p>
 +
<h3>Methods</h3>
 +
<p >This model simulates the kinetics of transcription signal the expression of DKK1 promoter. The process of protein synthesis is simulated with differential equations, assuming the synthesis of mCherry follows the central dogma of molecular biology. Signaling pathways of the androgen receptor is modeled based on chemical equilibrium of each transcription factor and intermediate.</p>
 +
<h3>Results</h3>
 +
<p> The increment of DKK1 gene expression rate caused by effective DHT activity </p>
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/a/ac/T--NYMU-Taipei--mCherryResult10.mov" autoplay="true" muted></video>
 +
<br>
 +
<p>Expected DKK1 protein expression level: [DKK1] = 5 + 0.006 [DHT]<sup>2</sup></p>
 +
<video loop="true" src="https://static.igem.org/mediawiki/2018/6/6f/T--NYMU-Taipei--FRETmCherryResult11.mov" autoplay="true" muted></video>
 +
<p> where [DKK1] indicates DKK1 activity in ng/ml and [DHT] indicates DHT activity in nM.</p>
 +
<p>Note: This is accurate only when [DHT] is less than 50 nM. [5]</p>
 +
<h3>Conclusion</h3>
 +
<p>[mCherry] = c<sub>1</sub> + c<sub>2</sub> [Testosterone]<sup>2</sup> </p>
 +
<p>where square brackets indicate activity in M, and c<sub>1</sub> and c<sub>2</sub> are constants to be determined.</p>
 +
<p>Note: This model is accurate only when [DHT] is less than 50 nM. [5]</p>
 +
<p class="detailtrigger" id="3t" onclick="detail(event)">Click Here For More Info</p>
 +
</div>
 +
<div class="detail story" id="3d">
 +
<embed src="https://static.igem.org/mediawiki/2018/0/0a/T--NYMU-Taipei--protocol-model-mCherry.pdf" style="width:800px; height:1200px; text-align:center;">
 +
</div>
 +
</div>
 +
 +
 +
<div class="story section">
 +
<h1 id="4" class="story-title subtitle">References</h1>
 +
<div class="paragraphs">
 +
<ol>
 +
<li>Victoria E. Ahn et al (2011). "Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6." Dev Cell . 2011 November 15; 21(5): 862–873.</li>
 +
<li>Zhihong Cheng (2011). "Crystal structures of the extracellular domain of LRP6 and its complex with DKK1." Nat Struct Mol Biol. 2011 Oct 9;18(11):1204-10.</li>
 +
<li>Jiménez-García B, Pons C, Fernández-Recio J. (2013). "pyDockWEB: a web server for rigid-body protein-protein docking using electrostatics and desolvation scoring." Bioinformatics. 2013 Jul 1;29(13):1698-9.</li>
 +
<li>Meehan KL1, Sadar MD. (2003). "Androgens and androgen receptor in prostate and ovarian malignancies." Front Biosci. 2003 May 1;8:d780-800.</li>
 +
<li>Kwack MH, Sung YK, Chung EJ, Im SU, Ahn JS, Kim MK, Kim JC. "Dihydrotestosterone-inducible dickkopf 1 from balding dermal papilla cells causes apoptosis in follicular keratinocytes." J Invest Dermatol. 2008 Feb;128(2):262-9.</li>
 +
</ol>
 +
</div>
 
</div>
 
</div>

Latest revision as of 01:36, 18 October 2018

HAIR TO STAY
TEAM
Team Members
Collaborations
PROJECT
Description
Design
Experiments & Results
Notebook
InterLab
Model
Demonstrate
Improve
Attributions
PARTS
Parts Overview
Basic Parts
Composite Parts
Part Collection
SAFETY
HUMAN PRACTICES
Human Practices
Education & Engagement
AWARDS
Applied Design
Entrepreneurship
Model
JUDGING FORM ⇗



FRET Ratio Model

Background

This model aims to find out how much FRET protein should be added into our screening system.

Methods

Chemical equilibrium is used to determine the concentration of each protein, the fluorescence level, and the minimal FRET protein activity required to produce the fluorescence that can be detected. This model assumes the portion of active protein in all proteins is constant. The equilibrium constants are from Victoria E. Ahn's study [1] and Zhihong Cheng's study [2]. The limited experiment budget for making FRET proteins is considered in this model.

Results

The relationship between fluorescence and DKK1 expression level

The relationship between fluorescence and the activity of one of the FRET proteins

The plot of the optimal activity of a FRET protein vs. the other

3D plot of the fluorescence, the objective function we want to maximize.

The x-axis is the concentration of one of the FRET protein and y-axis is that of the other FRET protein.

Contour a plot of the fluorescence, the objective function we want to maximize.

The x-axis is the concentration of one of the FRET protein and y-axis is that of the other FRET protein.

A plot of the optimal “cheaper FRET protein” activity, vs. budget

A plot of the optimal “more expensive FRET protein” activity, vs. budget

A plot of the optimal ratio vs. budget


Kd values obtained from the references:

Conclusion

The optimal ratio of the amount of one FRET protein to that of the other is as the following:

The figures in the table indicate the optimal ratio of the protein on the top over the protein on the left.

Click Here For More Info

FRET Efficiency Model

Background

The efficiency of FRET is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Therefore, simulating structure of protein-protein interactions is important. This model aims to predict the FRET efficiency in order to determine which molecules and which termini should be used.

Methods

pyDockWEB [3] is used for structural prediction of protein-protein interactions and the prediction of distances between donor and acceptor. Förster theory is used to determine the FRET efficiency. YPet and CyPet are added to either N-terminus or C-terminus of each of the 2 FRET proteins.

Results

The protein-protein interactions structural predictions are as follows:

Cyan: LRP6-BP1BP2      Green: LRP6-BP3BP4      Purple: DKK1


Cyan: LRP6-BP3BP4      Green: VHH G5      Purple: DKK1


Cyan: VHH H7      Green: VHH G5      Purple: DKK1

The predicted distances are

YPet and CyPet are added to the N-terminus or the C-terminus.


Conclusion

The FRET efficiency is given by the following table.

YPet and CyPet are added to the N-terminus or the C-terminus.


Click Here For More Info

The protein-protein interactions structural predictions can be downloaded from the following files
DKK1-E3E4G5.pse
DKK1E1E2E3E4.pse
DKK1E3E4.pdb
DKK1E3E4H7.pdb
DKK1G5H7-model-1.pse

mCherry Expression Model
















Background

This project constructs a plasmid that connects DKK1 promoter to mCherry. The gene expression rate of DKK1 promoter is affected by testosterone activity and affects the expression level of mCherry. The expression level of mCherry should be greater than a threshold so that its fluorescence can be detected by devices. In order to achieve this threshold, sufficient amount of testosterone should be added into our screening system; this is the sensitivity of our screening system. This model aims to find out the sensitivity of our screening system.

Methods

This model simulates the kinetics of transcription signal the expression of DKK1 promoter. The process of protein synthesis is simulated with differential equations, assuming the synthesis of mCherry follows the central dogma of molecular biology. Signaling pathways of the androgen receptor is modeled based on chemical equilibrium of each transcription factor and intermediate.

Results

The increment of DKK1 gene expression rate caused by effective DHT activity


Expected DKK1 protein expression level: [DKK1] = 5 + 0.006 [DHT]2

where [DKK1] indicates DKK1 activity in ng/ml and [DHT] indicates DHT activity in nM.

Note: This is accurate only when [DHT] is less than 50 nM. [5]

Conclusion

[mCherry] = c1 + c2 [Testosterone]2

where square brackets indicate activity in M, and c1 and c2 are constants to be determined.

Note: This model is accurate only when [DHT] is less than 50 nM. [5]

Click Here For More Info

References

  1. Victoria E. Ahn et al (2011). "Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6." Dev Cell . 2011 November 15; 21(5): 862–873.
  2. Zhihong Cheng (2011). "Crystal structures of the extracellular domain of LRP6 and its complex with DKK1." Nat Struct Mol Biol. 2011 Oct 9;18(11):1204-10.
  3. Jiménez-García B, Pons C, Fernández-Recio J. (2013). "pyDockWEB: a web server for rigid-body protein-protein docking using electrostatics and desolvation scoring." Bioinformatics. 2013 Jul 1;29(13):1698-9.
  4. Meehan KL1, Sadar MD. (2003). "Androgens and androgen receptor in prostate and ovarian malignancies." Front Biosci. 2003 May 1;8:d780-800.
  5. Kwack MH, Sung YK, Chung EJ, Im SU, Ahn JS, Kim MK, Kim JC. "Dihydrotestosterone-inducible dickkopf 1 from balding dermal papilla cells causes apoptosis in follicular keratinocytes." J Invest Dermatol. 2008 Feb;128(2):262-9.