Team:Imperial College London/Modelling/Protein Display

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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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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Surface Protein Model
 
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<div class="accordionButton">Objectives</div>
 
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The motivation to develop this model came from our design process. The idea of having surface proteins that could be cleaved and then activate the receptor was very innovatory. However, with this new approach questions arose that could not be answered without using computer models. Hence, the following aims for this model were specified:
 
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<ol>
 
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<li>Determine the concentration of Schistosoma elastase or TEV protease that should be added to the bacteria in order to trigger a response. This should allow us to correlate the required concentration for the activation with the concentration of Schistosoma elastase in the water.</li>
 
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<li>Attempt simulating how long it takes for the protease or elastase to cleave enough peptides.</li>
 
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<li>Assess the risk of false positives (i.e. system is activated without the required stimulus).</li>
 
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<div class="accordionButton">Detailed Description</div>
 
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This model consists of 5 parts that had to be developed:
 
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<ol>
 
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<li>Identification of all active and relevant elements of the isolated part of the system.</li>
 
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<li>Identification of interactions between the identified elements.</li>
 
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<li>Identification of threshold concentration of auto inducing peptide (AIP) needed for activation of the receptor.</li>
 
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<li>Defining a control volume around the bacterial cell (after cleavage, the surface protein will float around in the extracellular environment).</li>
 
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<li>Determining the surface protein production to estimate the maximum abundance on the bacterial surface.</li>
 
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<h2>1. Elements of the system</h2>
 
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<ol>
 
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<li>The surface protein consists of a cell wall binding domain, linker, AIP (Auto Inducing Peptide). It is expressed by constitutive gene expression. It was assumed that the bacteria would be fully grown before carrying out the detection so the cell wall would be covered with as many surface proteins as the cell can maintain.</li>
 
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<li>Schistosoma elastase (enzyme released by the parasite) cleaves the AIP from the cell wall binding domain at the linker site. In the laboratory we used TEV protease as we could not get hold of Schistosoma elastase, so the model was adjusted appropriately (TEV enzyme kinetic parameters were used).</li>
 
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<li>The ComD receptor is activated by a sufficiently high AIP concentration. Once activated, ComD signals into the cell to activate the colour response.</li>
 
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</ol>
 
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<h2>2. Interactions between elements </h2>
 
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Apart from the proteins being expressed from genes, there was only one more chemical reaction identified in this part of the system. This is the cleavage of proteins, which is an enzymatic reaction:
 
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<ul>
 
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<li>E+S <var>&harr;</var> ES <var>&rarr;</var> E+P</li>
 
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<li>Substrate (S) = Protein</li>
 
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<li>Enzyme (E) = TEV (Protease)</li>
 
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<li>Product (P) = Peptide</li>
 
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</ul><br />
 
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This enzymatic reaction can be rewritten as partial differential equations (PDEs), which is of similar form as the 1-step amplification model. However, most of the constants and initial concentrations are different. For detailed description and derivation of PDEs, please refer to <a href="https://2010.igem.org/Team:Imperial_College_London/Modelling/Output">"Detailed Description"</a> part of Modelling Output.<br />
 
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<h2>3. Threshold concentration of AIP</h2>
 
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The optimal peptide concentration required to activate ComD is 10 ng/ml <a href="http://ukpmc.ac.uk/backend/ptpmcrender.cgi?accid=PMC40587&blobtype=pdf">[1]</a>. This is the threshold value for ComD activation. However, the minimum concentration of peptide to give a detectable activation is 0.5ng/ml.
 
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<br />
 
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The threshold for the minimal activation of the receptor is c<sub>th</sub>=4.4658x10<sup>-9</sup> mol/L. Click on the button below to see the calculations.
 
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<div class="accordionButton">Converting 10 ng/ml to 4.4658x10<sup>-9</sup> mol/L</div>
 
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      <li>The mass of a peptide is 2.24kDa = 3.7184x10<sup>-21</sup>g.</li>
 
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      <li>The number of molecules in one ml is 10ng/3.7184x10<sup>-21</sup>g = 2.6893x10<sup>12</sup>. In a litre, the number of molecules is 2.6893x10<sup>15</sup>molecules/ml.</li>
 
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      <li>Dividing this value by Avogadro's constant gives the threshold concentration of c<sub>th</sub>=4.4658x10<sup>-9</sup> mol/L.</li>
 
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    <li>The threshold for minimal activation of receptor is 2.2329x10<sup>-10</sup> mol/L.</li>
 
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<h2>4. Control volume selection</h2>
 
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Note that this enzymatic reaction is modelled outside the cell. Hence, it is important to take into account the cell boundaries. It is worth considering whether diffusion or fluid movements will play a significant role.
 
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Initially, we defined a control volume assuming that bacteria would grow in close colonies on the plate. The details can be seen by clicking on the button below.
 
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<div class="accordionButton">Initial Choice of Control Volume</div>
 
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<b>Control volume initial choice</b><br/>
 
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This control volume is considered to be wrong, but the details were kept for the reference.<br/>
 
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The control volume:
 
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The inner boundary is determined by the bacterium (proteins that have been displayed and cleaved cannot diffuse back into the bacterium). The outer boundary is time scale dependent. We have assumed that after mass cleavage of the display proteins by TEV, many of these AIPs will bind to the receptors quickly (e.g. 8 seconds). Our volume is determined by the distance that the AIPs could travel by diffusion within that short time. In this way, we can ensure that the concentration of AIPs outside our control volume is approximately 0 (after a given time interval).
 
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This approach is not very accurate and can lead to false negative conclusions (as in reality there will be a concentration gradient, with the highest conentration on the cell wall).
 
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<img src="http://www.openwetware.org/images/4/42/Outer_Volume.JPG" width="600" alt="Control volume (Volume of B.sub to be excluded. x indicates the distance travelled by AIPs from the surface in time t)." />
 
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|Control volume (Volume of B.sub to be excluded.<br/> x indicates the distance travelled by AIPs from the surface in time t).
 
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Difussion distance was calculated using Fick's 1st Law: x=<var>&radic;</var>2Dt, where: x - diffusion distance, D - diffusion constant, t - time of diffusion
 
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<br />
 
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D<sub>average</sub> = 10.7x10<sup>-11</sup> m<sup>2</sup>s<sup>-1</sup> 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">[3]</a>
 
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t = 8s (arbitrarily chosen)
 
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This gives: x = 4.14x10<sup>-5</sup>m
 
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The control volume can be calculated by adding 2x to the length and the diamter of the cell.
 
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This gives a control volume (CV) = 4.81x10<sup>-7</sup>m<sup>3</sup>
 
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<p>We realized that our initial choice of control volume was not accurate because we had assumed that the bacteria was the medium. However, in reality bacteria live in colonies very close to each other. Since our bacteria was meant to be used in suspension we had to reconsider this issue.</p>
 
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<br/>
 
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<b>Using CFU to estimate the spacing between cells</b><br/>
 
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CFU stands for Colony-forming unit. It is a measure of bacterial numbers. For liquids, CFU is measured per ml.
 
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We already have data of CFU/ml from the Imperial iGEM 2008 team, so we could use this data to estimate the number of cells in a given volume using a spectrometer at 600nm wavelength.
 
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The graph below is taken from the Imperial iGEM 2008 Wiki page <a hred="https://2008.igem.org/Imperial_College/18_September_2008">[4]</a>.
 
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<img src="http://www.openwetware.org/images/b/bf/Calibration-Dilution.PNG" width="600" alt="CFU/ml vs. Optical Density at 600nm (OD600)." />
 
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|CFU/ml vs. Optical Density at 600nm (OD600).
 
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The graph shows values of CFU/ml for different optical densities. The range of CFU/ml is therefore between 0.5x10^<sup>8</sup> - 5x10<sup>8</sup>.
 
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In this calculation, we assumed that only one cell will grow and become one colony (i.e. no more than one cell will form no more than one colony). Therefore, the maximum number of cells in 1ml of solution is 5x10<sup>8</sup>. Taking the volume of 1 ml = 10<sup>-3</sup> dm<sup>3</sup> and dividing by the (maximum) number of cells in 1ml gives the average control volume (CV) around each cell: 2x10<sup>-12</sup> dm<sup>3</sup>/cell. For simplicity, we choose the control volume to be cubic. Taking the third root of this value gives the length of each side of the control volume.
 
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<p>Side length of CV = y = 1.26x10<sup>-4</sup> dm = 1.26x10<sup>-5</sup> m.</p>
 
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<b>Choice of Control Volume allows simplifications</b><br/>
 
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<ul>
 
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<li>Firstly, assume that the cells will be placed in the centre of the CV. Hence, after cleavage the protein will have an average distance of y/2 to travel in order to cross the boundary of the CV. This is calculated to happen within 0.18s. Even if the bacterium was not placed in the centre of the CV, the protein will travel from one end of the cube to the other in less than one second (~0.74s). Hence, it will take between 0.18 and 0.74s for the concentration of AIPs around the cell to be uniform. Noticing that these time values are very small, we can approximate our model to have a uniform concentration across the volume. Since we are underestimating the value of AIP concentration right next to the cell's surface, we are overestimating the time required for the AIP concentration to reach the threshold level.</li>
 
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<li>We can neglect the diffusive fluxes across the CV border (see figure below). Assuming that adjacent cells are producing the peptide at the same rate and that the concentration of TEV is the same around the cell, then the fluxes should be of the same value giving a net flux of zero. Hence, we can neglect diffusion and have our model limited to one bacterium.</li>
 
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<img src="http://www.openwetware.org/images/f/f9/Control_Volume_and_fluxes.JPG" width="600" alt="Figure showing 2 cells with their control volumes." />
 
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|Figure showing 2 cells with their control volumes.
 
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<br /><br />
 
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<b>Limitations resulting from our assumptions</b><br/>
 
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Most of our assumptions concerning the control volume were plausible due to the careful choice of cell density = 5x10<sup>8</sup> CFU/ml.
 
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<br />
 
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If the density is varied significantly, then our simplifications might not hold any longer. However, this does not mean that our system cannot function for lower cell densities. Our model might not be very accurate for predicting situations with cell densities that are much higher or lower than 5x10<sup>8</sup> CFU/ml.
 
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<br />
 
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It was decided that the model should not be used for cell densities lower 10<sup>7</sup> CFU/ml. Below this value AIPs take more than 1 second to diffuse accross half of a side-length of the control volume (assuming that the cell is inside control volume). We agreed that below 10^<sup>7</sup> CFU/ml the approximation about uniform concentration throughout the control volume could be wrong and that the concentration gradients could become more significant. If our model was applied to this particular situation, it would possibly overestimate the time taken to activate the receptor.
 
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<br />
 
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It is not possible to increase the cell density by more than 10<sup>9</sup> CFU/ml</math> because of the size of a cell.
 
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<h2>5. Protein production (23/08/2010)</h2>
 
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<ul>
 
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<li>The paper mentions 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">[2]</a></li>
 
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<li>2.4x10<sup>5</sup> molecules = 2.4x10<sup>5</sup>/6.02x10<sup>23</sup> mol = 0.398671x10<sup>-18</sup> mol</li>
 
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<li>Volume of B.sub: 2.79x10<sup>-15</sup> dm<sup>3</sup></math></li>
 
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<li>Concentration = [mol/L]</li>
 
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<li>c = 1.4289x10<sup>-4</sup> mol/dm<sup>3</sup>. This is the concentration of protein that will be displayed on a single cell of B.sub.
 
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</ul>
 
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<br />
 
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Hence, we can deduce that the final concentration that the protein expression will tend to is: c = 1.4289x10<sup>-4</sup> mol/dm<sup>3</sup> = c<sub>final</sub>.
 
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<br /><br />
 
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Therefore, we can model the protein production by transcription and translation and adjust the production constants so that the concentration will tend towards c<sub>final</sub>.
 
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<br />
 
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Using a similar model to the simple production of Dioxygenase for the Output Amplification Model (<a href="https://2010.igem.org/Team:Imperial_College_London/Modelling/Output">Model preA</a>), we obtain the following graph:
 
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<img src="http://www.openwetware.org/images/f/f5/Protein_production.jpg" width="600" alt="Production of protein by transcription and translation." />
 
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|Production of protein by transcription and translation.
 
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<p>The degradation rate was kept constant, and the production rate was changed according to the final concentration.</p>
 
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<b>Protein production in Control Volume</b><br/>
 
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The previously determined constants of protein production in B.sub to obtain the concentration of proteins are not valid in the Control Volume. It has to be adjusted (multiplied) by the following factor:
 
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factor=V<sub>bacillus</sub>/V<sub>CV</sub> = 5.7974x10<sup>-6</sup> (for the particular numbers presented above)
 
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<img src="http://www.openwetware.org/images/7/78/Protein_in_control_volume.jpg" width="600" alt="Production of protein by transcription and translation in control volume." />
 
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|Production of protein by transcription and translation in control volume.
 
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<h2>References</h2>
 
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<ol>
 
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<li>Havarstein, L., Coomaraswamy, G. & Morrison, D. (1995) An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. [Online] 92, 11140-11144. Available from: http://ukpmc.ac.uk/backend/ptpmcrender.cgi?accid=PMC40587&blobtype=pdf [Accessed 27th August 2010]</li>
 
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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>
 
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<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> 
 
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<li>Imperial College London (2008) Biofabricator Subtilis - Designer Genes. [Online] Available from: https://2008.igem.org/Imperial_College/18_September_2008 [Accessed 1st September 2010]</li>
 
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<h1>Constants for the Protein Display Model</h1>
 
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    <td style="background-color:#FFFF66;height:50px;width:75;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Type of Constant</b>
 
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    <td style="background-color:#FFFF99;height:50px;width:700;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Derivation of Value</b>
 
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    <td style="background-color:#FFCC66;height:100px;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>TEV Enzyme Dynamics</b>
 
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    <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">Enzymatic Reaction: E+S <var>&harr;</var> ES <var>&rarr;</var> E+P
 
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<br />
 
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The derivation of these values is made in <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Variables1">Variables for Amplification Module Section</a>.
 
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<li>k<sub>1</sub> = rate constant for E + S <var>&rarr;</var> ES = 10<sup>8</sup> M<sup>-1</sup>s<sup>-1</sup>
 
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<li>k<sub>2</sub> = rate constant for E + S <var>&larr;</var> ES = 10<sup>3</sup> s<sup>-1</sup>
 
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<li>k<sub>cat</sub> = rate constant for ES <var>&rarr;</var> E + P = 0.16 <var>&plusmn;</var> 0.01 s<sup>-1</sup>
 
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<br />
 
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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.
 
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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>
 
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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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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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<br />
 
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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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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>Degradation Rate of Surface Proteins (common for all)</b>
 
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    </td>
 
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    <td style="background-color:#e7e7e7;height:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;">Assumption: To be approximated by cell division (dilution of media) as none of the proteins are involved in any active degradation pathways.
 
-
<br />
 
-
Derived in <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Variables1">Variables for Amplification Module Section</a>:
 
-
<br />
 
-
k<sub>deg</sub>= 0.000289s<sup>-1</sup>
 
-
<br />
 
-
For all proteins that are outside of cells or the timescale that is short enough to neglect cell division effect: k<sub>deg</sub>=0
 
-
 
-
    </td>
 
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</tr>
 
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<tr>
 
-
    <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>
 
-
    <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>
 
-
<br />
 
-
In the final model the following was used:
 
-
For protein in water: D=10<sup>-10</sup>m<sup>2</sup>/s <a href="http://www.life.illinois.edu/crofts/bioph354/diffusion1.html">[3]</a>
 
-
    </td>
 
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<tr>
 
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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>
 
-
    <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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</table>
 
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<html>
 
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<h2>References</h2>
 
-
<ol>
 
-
<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>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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</ol>
 
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<div class="accordionButton">Results & Conclusions</div>
 
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<div class="accordionContent">
 
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{| style="background:#e7e7e7;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;margin- top:5px;" cellspacing="5";
 
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<img src="http://www.openwetware.org/images/7/77/Protein_display.png" width="600" alt="Graphs showing the simulation using [TEV]<sub>0</sub> = 4x10<sup>-4</sup> mol/dm<sup>3</sup>. The graph on the right hand-side below shows that the AIP threshold (red line) is reached after 22 s." />
 
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|Graphs showing the simulation using [TEV]<sub>0</sub> = 4x10<sup>-4</sup> mol/dm<sup>3</sup>.<br/> The graph on the right hand-side below shows that the AIP threshold (red line) is reached after 22 s.
 
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<html>
 
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<br/>
 
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<b>Sensitivity of our model (24/08/2010)</b></br><br/>
 
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<ul>
 
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<li>Changing initial concentration of TEV</li>
 
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Whether the threshold concentration of AIP is reached is highly dependent on the initial concentration of TEV. The smallest initial concentration of TEV, [TEV>]<sub0</sub>, for which the threshold is reached is 6.0x10<sup>-6</sup>mol/dm<sup>3</sup>. On the grap below it can be seen that the optimal [TEV]<sub>0</sub> is a concentration higher than 10<sup>-4</sup>mol/dm<sup>3</sup>, which corresponds to the threshold being reached within 1.5 minutes.
 
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</html>
 
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<div ALIGN=CENTER>
 
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{| style="background:#e7e7e7;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;margin- top:5px;" cellspacing="5";
 
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|-
 
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|<html>
 
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<img src="http://www.openwetware.org/images/d/df/AIP_Threshold_concentration.PNG" width="600" alt="Graph showing when threshold AIP concentration is reached (for different initial TEV concentrations). Notice log-log scale." />
 
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</html>
 
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|Graph showing when threshold AIP concentration is reached<br/> (for different initial TEV concentrations). Notice log-log scale.
 
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|}
 
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</div>
 
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<html><br/>
 
-
<li>Changing the production rate</li>
 
-
One order of magnitude change in the production rate results in at least 50s delay of the AIP concentration reaching the threshold concentration.<br/><br/>
 
-
<li>Changing production rate</li>
 
-
Changing the production rate influences the time duration of the AIP concentration above the threshold level. The higher it is, the shorter the receptor will be activated (at extreme values, AIP concentration does not reach the threshold). However, the production rate has not much influence on how fast the threshold will be reached.<br/><br/>
 
-
<li>Changing control volume</li>
 
-
Our model is extremely sensitive to this factor. One order of magnitude change in CV results in several orders of magnitude change in AIP concentration. Hence, special care should be taken in determination of this value. If the model is to be compared with the experimental results, the CFU/ml has to be the same as the one used in the model. Otherwise, the CV has to be readjusted.
 
-
</ul>
 
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<br/>
 
-
<b>Risk of False positives (31/08/2010)</b><br/>
 
-
It was pointed out that we should assess the risk of false positive activation of the receptor. We are particularly concerned  about the display protein not binding to the cell wall, but instead diffusing into the extra-cellular environment.
 
-
In order to be able to assess the risk of false positives, we need to do further research into the affinity of AIP with attached linker and transmembrane proteins for the receptor as compared to the affinity of the AIP itself for the receptor.
 
-
<br />
 
-
This paper <a href="http://jb.asm.org/cgi/content/full/186/10/3078">[1]</a> might have some information on affinity comparison.
 
-
We need to know how proteins are being transported from intracellular to transmembrane space. Understanding this concept could give us an idea of what could go wrong.
 
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<br/>
 
-
<h2>References</h2>
 
-
<ol>
 
-
<li>Knutsen, E., Ween, O. & Havarstein, L. (2003) Two Separate Quorum-Sensing Systems Upregulate Transcription of the Same ABC Transporter in Streptococcus pneumoniae. Journal of Bacteriology. [Online] 186(10), 3078-3085. Available from: http://jb.asm.org/cgi/reprint/186/10/3078 [Accessed 1st September 2010]</li>
 
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</ol>
 
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</div>
 
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<div class="accordionButton">Download: MatLab Files</div>
 
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<div class="accordionContent">
 
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<a href="https://static.igem.org/mediawiki/2010/5/52/Model_Protein_Display.txt"><img src="https://static.igem.org/mediawiki/2010/d/da/IC_Download4.png"></a>
 
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Latest revision as of 19:52, 17 October 2010