Team:Imperial College London/Modelling/Protein Display/Parameters and Constants

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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Parameters and Constants
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<a href="https://2010.igem.org/wiki/index.php?title=Team:Imperial_College_London/Modelling/Protein_Display/Objectives"><b>Objectives</b></a>; <a href="https://2010.igem.org/Team:Imperial_College_London/Modelling/Protein_Display/Detailed_Description"><b>Detailed Description</b></a>; <a href="https://2010.igem.org/Team:Imperial_College_London/Modelling/Protein_Display/Parameters_and_Constants"><b>Parameters & Constants</b></a>; <a href="https://2010.igem.org/Team:Imperial_College_London/Modelling/Protein_Display/Results_and_Conclusion"><b>Results & Conclusion</b></a>;<a href="https://2010.igem.org/Team:Imperial_College_London/Modelling/Protein_Display/Download_MatLab_Files"><b>Download MatLab Files</b></a>;
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<h2>Parameters & Constants</h2>
 
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     <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Production Rate of Surface Proteins</b>
     <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Production Rate of Surface Proteins</b>
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     <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">It was found that each cell displays 2.4x10<sup>5</sup> peptides <a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2000.tb09188.x/pdf">[1]</a>.  
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     <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">It was found that each cell displays 2.4&times;10<sup>5</sup> peptides <a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2000.tb09188.x/pdf">[1]</a>.  
Hence, we adjusted our simple production of display protein model to converge to that value. As production rate was the constant that we could not obtain, that value was manipulated.
Hence, we adjusted our simple production of display protein model to converge to that value. As production rate was the constant that we could not obtain, that value was manipulated.
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The result 4.13x10<sup>-8</sup>mol/dm<sup>3</sup>/s seemed to be of reasonable order of magnitude.
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The result 4.13&times;10<sup>-8</sup>mol/dm<sup>3</sup>/s seemed to be of reasonable order of magnitude.
Ideally, we would like to get this value measured as it is resulting from a very vague estimate.
Ideally, we would like to get this value measured as it is resulting from a very vague estimate.
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     <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Diffusion Coefficient of Proteins</b>
     <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Diffusion Coefficient of Proteins</b>
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     <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">We have found two references which quote very similar values for very different media.<br />For protein in agarose gel: D<sub>average</sub> = 1.07x10<sup>-10</sup>m<sup>2</sup>/s - for a protein in agarose gel for pH=5.6 <a href="http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6V5N-4B3MXDC-2-K&_cdi=5791&_user=217827&_pii=S1369703X03002377&_origin=search&_coverDate=07%2F01%2F2004&_sk=999809998&view=c&wchp=dGLzVtb-zSkzS&md5=c17d0e7320f03931006f9b1a10a438b9&ie=/sdarticle.pdf">[2]</a>
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     <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">We have found two references which quote very similar values for very different media.<br />For protein in agarose gel: D<sub>average</sub> = 1.07&times;10<sup>-10</sup>m<sup>2</sup>/s - for a protein in agarose gel for pH=5.6 <a href="http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6V5N-4B3MXDC-2-K&_cdi=5791&_user=217827&_pii=S1369703X03002377&_origin=search&_coverDate=07%2F01%2F2004&_sk=999809998&view=c&wchp=dGLzVtb-zSkzS&md5=c17d0e7320f03931006f9b1a10a438b9&ie=/sdarticle.pdf">[2]</a>
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In the final model the following was used:
In the final model the following was used:
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     <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Control Volume</b>
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     <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Localised Concentration Coefficient</b>
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     <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">The control volume seems to be the weakest point of this model. We have tried to rationalise it as much as we could. However, errors seem to be unavoidable. It is important to realise that the Control Volume needs to be adjusted if a bacterial concentration different than 5x10<sup>8</sup>CFU/ml is used.
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     <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">The localised concentration coefficient seems to be the weakest point of this model. We have tried to rationalise it as much as we could. However, errors seem to be unavoidable. It is important to realise that the value of localised concentration coefficient probably would require adjusting for different bacterial concentrations.
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    <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Cell Wall thickness</b>
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    <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"> The cell wall thickness was found to be around 35nm. <a href="http://jb.asm.org/cgi/reprint/176/5/1413?ijkey=27dafbac7e23dee50390d3fe67d9d1bab0c6f48c">[5]</a>:
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    <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Diffusion constant</b>
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    <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"> The diffusion coefficient of small protein in water is of order = 10<sup>-6</sup> cm<sup>2</sup> s<sup>-1</sup> <a href="http://www.life.illinois.edu/crofts/bioph354/diffusion1.html">[6]</a>:
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<h2>References</h2>
 
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<li>Kobayashi, G. et al (2000) Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants. FEMS Microbiology Letters. [Online] 188(2000), 165-169. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2000.tb09188.x/pdf [Accessed 27th August 2010]</li>
<li>Kobayashi, G. et al (2000) Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants. FEMS Microbiology Letters. [Online] 188(2000), 165-169. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2000.tb09188.x/pdf [Accessed 27th August 2010]</li>
<li>Gutenwik, J., Nilsson, B. & Axelsson, A. (2003) Determination of protein diffusion coefficients in agarose gel with a diffusion cell. Biochemical Engineering Journal. [Online] 19(2004), 1-7. Available from: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6V5N-4B3MXDC-2-K&_cdi=5791&_user=217827&_pii=S1369703X03002377&_origin=search&_coverDate=07%2F01%2F2004&_sk=999809998&view=c&wchp=dGLzVtb-zSkzS&md5=c17d0e7320f03931006f9b1a10a438b9&ie=/sdarticle.pdf [Accessed August 20th 2010]</li>
<li>Gutenwik, J., Nilsson, B. & Axelsson, A. (2003) Determination of protein diffusion coefficients in agarose gel with a diffusion cell. Biochemical Engineering Journal. [Online] 19(2004), 1-7. Available from: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6V5N-4B3MXDC-2-K&_cdi=5791&_user=217827&_pii=S1369703X03002377&_origin=search&_coverDate=07%2F01%2F2004&_sk=999809998&view=c&wchp=dGLzVtb-zSkzS&md5=c17d0e7320f03931006f9b1a10a438b9&ie=/sdarticle.pdf [Accessed August 20th 2010]</li>
<li>Crofts, A. (1996) Biophysics 345. [Online] Available from: http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed 1st September 2010]</li>
<li>Crofts, A. (1996) Biophysics 345. [Online] Available from: http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed 1st September 2010]</li>
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<li>BioNumbers is the on-line database storing values of biologically related parameters with references; Available from: Available from: http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed 1st September 2010] [Accessed 19th August 2010]</li>
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<li>BioNumbers is the on-line database storing values of biologically related parameters with references; [Online] Available from: http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed August 19th 2010]</li>
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<li>Graham L. L. & Beverisge T. J. (1993) Structural Differentiation of the Bacillus subtilis 168 Cell Wall. Journal of Bacteriolofy. [Online] 5(1994), 1413-1420. Available from: http://jb.asm.org/cgi/reprint/176/5/1413?ijkey=27dafbac7e23dee50390d3fe67d9d1bab0c6f48c [Accessed October 26th 2010]</li>
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<li>Value was presented at Home Page for the Crofts laboratory (A. R. Crofts), at the University of Illinois at Urbana-Champaign; [Online] Available from:  http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed September 15th 2010]</li>
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Latest revision as of 22:57, 27 October 2010

Modelling Overview | Detection Model | Signaling Model | Fast Response Model | Interactions
A major part of the project consisted of modelling each module. This enabled us to decide which ideas we should implement. Look at the Fast Response page for a great example of how modelling has made a major impact on our design!
Objectives | Description | Results | Constants | MATLAB Code
Parameters and Constants

Type of Constant Derivation of Value
TEV Enzyme Dynamics Enzymatic Reaction: E+S ↔ ES → E+P
  • k1 = rate constant for E + S → ES = 108 M-1s-1
  • k2 = rate constant for E + S ← ES = 103 s-1
  • kcat = rate constant for ES → E + P = 0.16 ± 0.01 s-1

We are assuming the same cleaving rates of TEV as on other substrates. However, we are planning to measure them to gain more confidence in the model.
Production Rate of Surface Proteins It was found that each cell displays 2.4×105 peptides [1]. Hence, we adjusted our simple production of display protein model to converge to that value. As production rate was the constant that we could not obtain, that value was manipulated.
The result 4.13×10-8mol/dm3/s seemed to be of reasonable order of magnitude. Ideally, we would like to get this value measured as it is resulting from a very vague estimate.
Degradation Rate of Surface Proteins (common for all) Assumption: To be approximated by cell division (dilution of media) as none of the proteins are involved in any active degradation pathways.
kdeg= 0.000289s-1
For all proteins that are outside of cells or the timescale that is short enough to neglect cell division effect: kdeg=0
Diffusion Coefficient of Proteins We have found two references which quote very similar values for very different media.
For protein in agarose gel: Daverage = 1.07×10-10m2/s - for a protein in agarose gel for pH=5.6 [2]
In the final model the following was used: For protein in water: D=10-10m2/s [3]
Localised Concentration Coefficient The localised concentration coefficient seems to be the weakest point of this model. We have tried to rationalise it as much as we could. However, errors seem to be unavoidable. It is important to realise that the value of localised concentration coefficient probably would require adjusting for different bacterial concentrations.
Cell Dimensions Bacillus subtillis was treated as a rod unless specifically state otherwise.
The following dimensions were used [4]:
  • Diameter: 0.87 μm
  • Length: 4.7 μm
Cell Wall thickness The cell wall thickness was found to be around 35nm. [5]:
Diffusion constant The diffusion coefficient of small protein in water is of order = 10-6 cm2 s-1 [6]:

Click here to download the MATLAB code for this model...
References

  1. Kobayashi, G. et al (2000) Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants. FEMS Microbiology Letters. [Online] 188(2000), 165-169. Available from: http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2000.tb09188.x/pdf [Accessed 27th August 2010]
  2. Gutenwik, J., Nilsson, B. & Axelsson, A. (2003) Determination of protein diffusion coefficients in agarose gel with a diffusion cell. Biochemical Engineering Journal. [Online] 19(2004), 1-7. Available from: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6V5N-4B3MXDC-2-K&_cdi=5791&_user=217827&_pii=S1369703X03002377&_origin=search&_coverDate=07%2F01%2F2004&_sk=999809998&view=c&wchp=dGLzVtb-zSkzS&md5=c17d0e7320f03931006f9b1a10a438b9&ie=/sdarticle.pdf [Accessed August 20th 2010]
  3. Crofts, A. (1996) Biophysics 345. [Online] Available from: http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed 1st September 2010]
  4. BioNumbers is the on-line database storing values of biologically related parameters with references; [Online] Available from: http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed August 19th 2010]
  5. Graham L. L. & Beverisge T. J. (1993) Structural Differentiation of the Bacillus subtilis 168 Cell Wall. Journal of Bacteriolofy. [Online] 5(1994), 1413-1420. Available from: http://jb.asm.org/cgi/reprint/176/5/1413?ijkey=27dafbac7e23dee50390d3fe67d9d1bab0c6f48c [Accessed October 26th 2010]
  6. Value was presented at Home Page for the Crofts laboratory (A. R. Crofts), at the University of Illinois at Urbana-Champaign; [Online] Available from: http://www.life.illinois.edu/crofts/bioph354/diffusion1.html [Accessed September 15th 2010]

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