Difference between revisions of "Team:Aalto-Helsinki/Modeling propane"
(sidenav doesn't appear on small devices) |
(texts, mainly on sensitivity analysis) |
||
Line 63: | Line 63: | ||
<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 could 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> | <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 could 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> | ||
+ | <!-- Introduction above --> | ||
+ | |||
+ | <!-- Materials and methods section below --> | ||
<section id="mm" data-anchor="mm"> | <section id="mm" data-anchor="mm"> | ||
− | <h2>Materials and methods: Building the model | + | <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 basic 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>We built a model of our propane pathway based on Michaelis-Menten enzyme kinetics. It is a basic 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> | ||
Line 77: | Line 80: | ||
<p>For modeling this propane pathway we used <a href="http://www.copasi.org/tiki-view_articles.php">Copasi</a> and <a href="http://se.mathworks.com/">Matlab</a>.</p> | <p>For modeling this propane pathway we used <a href="http://www.copasi.org/tiki-view_articles.php">Copasi</a> and <a href="http://se.mathworks.com/">Matlab</a>.</p> | ||
</section> | </section> | ||
+ | <!-- Materials and methods above --> | ||
+ | |||
+ | <!-- Results section above --> | ||
<section id="results" data-anchor="results"> | <section id="results" data-anchor="results"> | ||
<h2>Results and implications</h2> | <h2>Results and implications</h2> | ||
− | < | + | <p>Our files for upload: <a href="https://static.igem.org/mediawiki/2015/8/85/Aalto-Helsinki_propane_pathway_Hbd.zip">Copasi file with Hbd</a>, <a href="https://static.igem.org/mediawiki/2015/c/ca/Aalto-Helsinki_propane_pathway_fadB2.zip">copasi file with FadB2</a> and our <a href="">Matlab file</a>.</p> |
+ | |||
+ | <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 our Car is mostly in its active form so we have assumed that it is all activated in following calculations.</p> | <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 our Car is mostly in its active form so 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 know which 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 code to plot these with Matlab | + | <p>To know which 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="">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> | ||
− | <figure> | + | <figure id="fig2"> |
<div style="width:80%;margin-left:auto;margin-right:auto;"><img src="https://static.igem.org/mediawiki/2015/d/dd/Aalto-Helsinki_Michaelis_plots.png" style="max-width:100%;" /></div> | <div style="width:80%;margin-left:auto;margin-right:auto;"><img src="https://static.igem.org/mediawiki/2015/d/dd/Aalto-Helsinki_Michaelis_plots.png" style="max-width:100%;" /></div> | ||
− | <figcaption><b>Figure | + | <figcaption><b>Figure 2:</b> Michaelis-Menten reaction rate plots for our enzymes.</figcaption> |
</figure> | </figure> | ||
− | < | + | <p style="color:red">Here be more beautiful pictures of bottlenecks</p> |
− | < | + | |
− | <p>The results shown in figure | + | <p>The results shown in <a href="#fig2">figure 2</a> tell us that FadB2 is a really bad enzyme and quite a large bottleneck in our reaction. This find 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> | ||
Line 106: | Line 114: | ||
<p>Car isn’t the best enzyme in our pathway, and unfortunately we couldn’t do anything to make it’s performance better because it was in different construct than Ado. We had ordered our constructs before we knew the bottleneck results and because of time restrictions we had to cope with what we had, but based on the obtained results we can deduce a better ordering of constructs than we now have. To the higher copy number backbone we should put as many of the slowest enzymes as possible.</p> | <p>Car isn’t the best enzyme in our pathway, and unfortunately we couldn’t do anything to make it’s performance better because it was in different construct than Ado. We had ordered our constructs before we knew the bottleneck results and because of time restrictions we had to cope with what we had, but based on the obtained results we can deduce a better ordering of constructs than we now have. To the higher copy number backbone we should put as many of the slowest enzymes as possible.</p> | ||
− | <p>We could also confirm these results by checking the fluxes through reactions and running parameter scan for different enzymes with Copasi | + | <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 figure | + | <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 real information 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> | + | <figure id="fig4"> |
<div style="width:80%;margin-left:auto;margin-right:auto;"><img src="https://static.igem.org/mediawiki/2015/b/ba/Aalto-Helsinki_Michaelis_plots_varying_enzymes.png" style="max-width:100%;" /></div> | <div style="width:80%;margin-left:auto;margin-right:auto;"><img src="https://static.igem.org/mediawiki/2015/b/ba/Aalto-Helsinki_Michaelis_plots_varying_enzymes.png" style="max-width:100%;" /></div> | ||
− | <figcaption><b>Figure | + | <figcaption><b>Figure 4:</b> Michaelis-Menten reaction rate plots with different enzyme concentrations based on the backbone copy numbers</figcaption> |
</figure> | </figure> | ||
</section> | </section> | ||
+ | |||
<section id="sensitivity" data-anchor="sensitivity"> | <section id="sensitivity" data-anchor="sensitivity"> | ||
<h3 >Sensitivity analysis</h3> | <h3 >Sensitivity analysis</h3> | ||
− | <p>We performed sensitivity analysis of our pathway model to see the robustness of different parameters. We performed this analysis with the aid of Copasi, which has a ready task for it. Further, we performed this analysis based on both parameters | + | <p>We performed sensitivity analysis of our pathway model to see the robustness of different parameters. We performed this analysis with the aid of Copasi, which has a ready task for it. Further, we performed this analysis based on both parameters and initial concentrations. </p> |
− | <p | + | <p>When we calculated the scaled sensitivities in regarding to initial concentrations, Butyryl-CoA was the most sensitive species of our reaction pathway. Hbd and NADPH influenced it positively (5.14 and 1.89) and YciA had a big negative influence (5.1). The results weren't very surprising; Hbd being one of our bottlenecks. From the results we could also see that propane was positively sensitive to Ado, Car, Hbd and NADPH. This confirms the main bottlenecks and also suggests that NADPH affects our propane production. To improve the pathway in future one could add mechanisms to create it in the cell higher amounts.</p> |
− | <p> | + | <p style="font-size:13px;">Here you can download the <a href="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_sensitivity_initConcentrations.txt">sensitivity results for initial concentrations</a> or our whole <a href="https://static.igem.org/mediawiki/2015/8/85/Aalto-Helsinki_propane_pathway_Hbd.zip">copasi file with Hbd</a>.</p> |
+ | |||
+ | <p>The relative sensitivities when calculated based on the parameters (i.e kinetic constants) tell us the familiar tale of Hbd, Car and Ado being the most influential enzymes in our pathway. The greatest single sensitivity (5.14) was again for Butyryl-Coa and caused by Hbd's \(K_{cat}\). YciA's \(K_{cat}\) has the smallest sensitivity value (-5.1). The results suggest that we could improve propane production by finding substitutive enzymes with better kinetic constants and performances.</p> | ||
+ | |||
+ | <p style="font-size:13px;">Here you can download the <a href="https://static.igem.org/mediawiki/2015/3/38/Aalto-Helsinki_sensitivity_parameters.txt">sensitivity results for parameters</a> or our whole <a href="https://static.igem.org/mediawiki/2015/8/85/Aalto-Helsinki_propane_pathway_Hbd.zip">copasi file with Hbd</a>.</p> | ||
− | |||
− | |||
<h3 id="timecourse">Time course</h3> | <h3 id="timecourse">Time course</h3> | ||
− | <p>From time course analysis we can have some kind of idea how much propane our system is able to produce. We performed this analysis with Copasi. Since we knew from the bottleneck calculations that FadB2 should be changed to Hdb, we considered only the latter | + | <p>From time course analysis we can have some kind of idea how much propane our system is able to produce. We performed this analysis with Copasi. Since we knew from the bottleneck calculations that FadB2 should be changed to Hdb, we considered only the latter. Also we had different amounts of enzymes based on which backbone they were.</p> |
− | <p>In our | + | <p>In our <a href="https://static.igem.org/mediawiki/2015/8/85/Aalto-Helsinki_propane_pathway_Hbd.zip">copasi file</a> we don't have the competing enzymes that also eat Butyraldehyde, the last substrate before Propane. This means that the values obtained here might be higher than in reality. <span style="color:red">Reference to particle model?</span></p> |
<p>With step length of 0.1 minutes and total time 100 min, we got for propane concentration 6.8e-07 mol/l.</p> | <p>With step length of 0.1 minutes and total time 100 min, we got for propane concentration 6.8e-07 mol/l.</p> | ||
Line 142: | Line 153: | ||
<p>The predicted amount of propane produced isn't very high. To produce propane more efficiently in the future, we would need to greatly improve the performance of our pathway. This could be done by taking a look into bottlenecks of our pathway and other limiting factors such as NADPH.</p> | <p>The predicted amount of propane produced isn't very high. To produce propane more efficiently in the future, we would need to greatly improve the performance of our pathway. This could be done by taking a look into bottlenecks of our pathway and other limiting factors such as NADPH.</p> | ||
</section> | </section> | ||
+ | <!-- Results section ends --> | ||
+ | |||
+ | <!-- Discussion section begins --> | ||
<section id="discussion" data-anchor="discussion"> | <section id="discussion" data-anchor="discussion"> | ||
<h2>Discussion</h2> | <h2>Discussion</h2> |
Revision as of 10:19, 26 August 2015