Difference between revisions of "Team:Aalto-Helsinki/Modeling propane"
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<h2 id="bottlenecks">Bottlenecks: Comparing enzyme rates</h2> | <h2 id="bottlenecks">Bottlenecks: Comparing enzyme rates</h2> | ||
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+ | <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 obtained Michaelis-Menten equations tell us directly the reaction speeds. We implemented the code to plot these with Matlab, it is available here(<- download link).</p> | ||
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+ | <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:gray">--pic of results here--</p> | ||
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+ | <p>The results shown above tell us that FadB2 is 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> | ||
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+ | <p>The plot also shows us that Ado is really bad enzyme, even though we use the mutated version with better performance than the original one (link to this?). To ease Ado-bottleneck, we put the construct containing it to the backbone that had higher copy number.</p> | ||
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+ | <p>Car isn’t very good enzyme either, 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.</p> | ||
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+ | <p>We could also confirm the results checking the fluxes through reactions and running parameter scan for different enzymes with Copasi. After identifying one bottleneck this way removed that enzyme from our model of the reaction pathway and repeating the calculations.</p> | ||
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+ | <p>Based on the results we can further deduce 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> | ||
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+ | <p>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 the different copy numbers of different backbones. It is good to remember that we don’t have real information how much there are enzymes in the cell so the actual values of these might not be right. Despite that this approach gives us good idea how one could improve the pathway in the future.</p> | ||
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+ | <p style="color:gray">--pic here--</p> | ||
<h2 id="timecourse">Time course</h2> | <h2 id="timecourse">Time course</h2> |
Revision as of 10:42, 5 August 2015