Difference between revisions of "Team:Aalto-Helsinki/Modeling propane"
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<p>Propane is a commonly used, convenient and clean-burning fuel, currently produced from non-renewable sources. Our project is about producing propane in bacteria, paving way for its sustainable production from renewable biomass. Ultimately, the pathway could be transferred to cyanobacteria, producing propane from CO2 and solar energy. </p> | <p>Propane is a commonly used, convenient and clean-burning fuel, currently produced from non-renewable sources. Our project is about producing propane in bacteria, paving way for its sustainable production from renewable biomass. Ultimately, the pathway could be transferred to cyanobacteria, producing propane from CO2 and solar energy. </p> | ||
− | <p> In our mathematical model our goal is to grasp the important concepts underlying the experiments made in the lab, and to see how those concepts could help us produce more propane. By having a better understanding of the ideas that govern our project, we | + | <p> In our mathematical model our goal is to grasp the important concepts underlying the experiments made in the lab, and to see how those concepts could help us produce more propane. By having a better understanding of the ideas that govern our project, we can see the influence of each compound in the reaction pathway and have a basis to make decisions that would have a long term impact in our results.</p> |
</section> | </section> | ||
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<h2>Materials and methods: Building the model</h2> | <h2>Materials and methods: Building the model</h2> | ||
− | <p>We built a model of our propane pathway based on Michaelis-Menten enzyme kinetics. It is a | + | <p>We built a model of our propane pathway based on Michaelis-Menten enzyme kinetics. It is a way to model enzyme reactions that assumes that the change that enzyme causes is faster than the binding of the enzyme and releasing of the substrate.</p> |
<p style="color:gray">--picture of pathway somewhere here--</p> | <p style="color:gray">--picture of pathway somewhere here--</p> | ||
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<h3 id="caractiv">Car-activation</h3> | <h3 id="caractiv">Car-activation</h3> | ||
− | <p>One of the enzymes in our pathway, Car, needs activation before it can function. To further understand how this affects the function of this enzyme we <a href="https://2015.igem.org/Team:Aalto-Helsinki/Car-activation">modeled the reactions governing the activation</a>. To summarize the results: in most scenarios | + | <p>One of the enzymes in our pathway, Car, needs activation before it can function. To further understand how this affects the function of this enzyme we <a href="https://2015.igem.org/Team:Aalto-Helsinki/Car-activation">modeled the reactions governing the activation</a>. To summarize the results: in most scenarios Car is mostly in its active form. This is why we have assumed that it is all activated in following calculations.</p> |
<h3 id="bottlenecks">Bottlenecks: Comparing enzyme rates</h3> | <h3 id="bottlenecks">Bottlenecks: Comparing enzyme rates</h3> | ||
− | <p>To | + | <p>To find out what are the rate limiting steps in our pathway, we compared the rates of the enzyme reactions. This was done by calculating the reaction speeds with different substrate concentrations. The reactions are explained in depth <a href="https://2015.igem.org/Team:Aalto-Helsinki/Kinetics">here</a> and the estimated Michaelis-Menten rate equations tell us directly the reaction speeds. We implemented the <a href="https://static.igem.org/mediawiki/2015/e/ed/Aalto-Helsinki_bottleneck_plots.m" download>code to plot these with Matlab</a>.</p> |
<p>FadB2 reaction is reversible in our model but for this we approximated it as irreversible. This yields better results for it than in reality.</p> | <p>FadB2 reaction is reversible in our model but for this we approximated it as irreversible. This yields better results for it than in reality.</p> | ||
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<p style="color:red">Here be more beautiful pictures of bottlenecks</p> | <p style="color:red">Here be more beautiful pictures of bottlenecks</p> | ||
− | <p>The results shown in <a href="#fig2">figure 2</a> tell us that FadB2 is a really | + | <p>The results shown in <a href="#fig2">figure 2</a> tell us that FadB2 is a really inefficient enzyme and one of the largest bottlenecks in our pathway. This caused us to change it to Hdb; an enzyme with same function and reportedly better performance.</p> |
<p>The plot also shows us that Ado isn't a good one either. To ease Ado-bottleneck, we put the construct containing it to the backbone that had higher copy number.</p> | <p>The plot also shows us that Ado isn't a good one either. To ease Ado-bottleneck, we put the construct containing it to the backbone that had higher copy number.</p> | ||
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<p>We could also confirm these results by checking the fluxes through reactions and running parameter scan for different enzymes with Copasi. After identifying one bottleneck this way we removed that enzyme from our model of the reaction pathway and repeated the calculations.</p> | <p>We could also confirm these results by checking the fluxes through reactions and running parameter scan for different enzymes with Copasi. After identifying one bottleneck this way we removed that enzyme from our model of the reaction pathway and repeated the calculations.</p> | ||
− | <p style="padding-top:1%;">After getting these results we performed the bottleneck analysis again out of curiosity with relative enzyme amounts. When before we had all the enzyme concentrations to be 1e-6 mol/l, now we scaled them to correspond to the different copy numbers of different backbones. We had put Car-construct into pSB6A1 (ORI: pMB1, copynumber: 15-20) and Ado-construct into pCDFDuet-1 (ORI: CloDF13, copynumber: 20-40). Based on this we approximated that there is about 1.5 times more of those enzymes that are in Ado construct; see <a href="#fig4">figure 4</a> for results. It is good to remember that we don’t have | + | <p style="padding-top:1%;">After getting these results we performed the bottleneck analysis again out of curiosity with relative enzyme amounts. When before we had all the enzyme concentrations to be 1e-6 mol/l, now we scaled them to correspond to the different copy numbers of different backbones. We had put Car-construct into pSB6A1 (ORI: pMB1, copynumber: 15-20) and Ado-construct into pCDFDuet-1 (ORI: CloDF13, copynumber: 20-40). Based on this we approximated that there is about 1.5 times more of those enzymes that are in Ado construct; see <a href="#fig4">figure 4</a> for results. It is good to remember that we don’t have accurate information on how much there are enzymes in the cell so the actual values might not be right. Despite that this approach gives us a good idea of how one could improve the pathway in the future.</p> |
<figure id="fig4"> | <figure id="fig4"> |
Revision as of 07:15, 31 August 2015