Difference between revisions of "Team:NYMU-Taipei/Model"
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<p >The optimal ratio of the amount of one FRET protein to that of the other is as the following:</p> | <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/2018/9/9d/T--NYMU-Taipei--FRETRatioConclusion11.png"> | <img src="https://static.igem.org/mediawiki/2018/9/9d/T--NYMU-Taipei--FRETRatioConclusion11.png"> | ||
| − | <p >The figures in the table | + | <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> | ||
</div> | </div> | ||
Revision as of 14:00, 17 October 2018
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 protein is constant. The equilibrium constants are from Victoria E. Ahn's study [1] and Zhihong Cheng's study [2]. The experiment budget for 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. another
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
Contour 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
Plot of the optimal “cheaper FRET protein” activity, vs. budget
Plot of the optimal “more expensive FRET protein” activity, vs. budget
Plot of the optimal ratio vs. budget
K_d 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 terminals 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 determine the FRET efficiency.
Results
The protein-protein interactions structural predictions are as follows:
The predicted distances are (Ångström)
where N means YPet or CyPet is added to the N terminal and C means YPet or CyPet is added to the C terminal.
Conclusion
The FRET efficiency is given by the following table.
where N means YPet or CyPet is added to the N terminal and C means YPet or CyPet is added to the C terminal.
Click Here For More Info
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
[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.
Conclusion
[mCherry] = c_1 + c_2 [Testosterone]^2
where square brackets indicate activity in M, and c_1 and c_2 are constants to be determined.
Note: This model is accurate only when [DHT] is less than 50 nM.
Click Here For More Info
References
- 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. doi:10.1016/j.devcel.2011.09.003.
- Zhihong Cheng (2011). "Crystal structures of the extracellular domain of LRP6 and its complex with DKK1." VOLUME 18 NUMBER 11 NOVEMBER 2011 nature structural & molecular biology.
- 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. doi: 10.1093/bioinformatics/btt262. Epub 2013 May 9.
- Meehan KL1, Sadar MD. (2003). "Androgens and androgen receptor in prostate and ovarian malignancies." Front Biosci. 2003 May 1;8:d780-800.