Difference between revisions of "Team:Aalto-Helsinki/Results"
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<h3>Background</h3> | <h3>Background</h3> | ||
− | <p>Microbially produced propane holds enormous promise as a potential replacement of portable fossil fuels, but the propane yields with current biological pathways are low. The pathway | + | <p>Microbially produced propane holds enormous promise as a potential replacement of portable fossil fuels, but the propane yields with current biological pathways are low. The pathway starts from acetyl-CoA and produces propane through 6 intermediates. To help concentrate engineering efforts on its critical parts, better quantitative understanding of the pathway is required. Our goals were to build a mathematical model of the pathway to better understand it and create and test BioBricks of the propane pathway to help future teams and researchers to continue improving it.</p> |
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<figure style="float:right;margin-left:20px;margin-bottom:3%;"> | <figure style="float:right;margin-left:20px;margin-bottom:3%;"> | ||
<img src="https://static.igem.org/mediawiki/2015/a/aa/Aalto-Helsinki_bottleneck_both.png" style="width:400px;"/> | <img src="https://static.igem.org/mediawiki/2015/a/aa/Aalto-Helsinki_bottleneck_both.png" style="width:400px;"/> | ||
− | <figcaption><b>Figure 1:</b> Illustrative figure of the bottleneck results of our pathway.</figcaption> | + | <figcaption><b>Figure 1:</b> Illustrative figure of the bottleneck results of our pathway.On the left,</br>situation before switching FadB2 to Hbd, on the right situation after the switch.</figcaption> |
</figure> | </figure> | ||
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<li><p>Improved our experimental plans according to the modeling results by changing one enzyme to a better homolog and expressing the rate-limiting enzyme from the highest copy number backbone used</p></li> | <li><p>Improved our experimental plans according to the modeling results by changing one enzyme to a better homolog and expressing the rate-limiting enzyme from the highest copy number backbone used</p></li> | ||
<li><p>Found that propane output was sensitive to NADPH/NADH, suggesting their efficient regeneration might be a limiting factor</p></li> | <li><p>Found that propane output was sensitive to NADPH/NADH, suggesting their efficient regeneration might be a limiting factor</p></li> | ||
− | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts#propane_1">Submitted a BioBrick</a> containing three crucial enzymes of the propane pathway</p></li> | + | <li><p>Designed two BioBricks containing all ten genes required for the propane pathway to produce propane in E. coli |
− | + | <ul> | |
+ | <li><p>Chose three intercompatible backbones to ensure that the propane pathway could be integrated to the same cell with cellulose hydrolysis (which required the third backbone)</p></li> | ||
+ | </ul></p></li> | ||
+ | <figure style="float:right;margin-left:20px;margin-top:20px;margin-bottom:20px;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/3/3a/Aalto-Helsinki_car_submittedparts.png" style="width:315px;"/> | ||
+ | <figcaption>Assembled Propane 1 (CAR) constructs.</figcaption> | ||
+ | </figure> | ||
+ | |||
+ | <figure style="float:right;margin-left:20px;margin-top:20px;margin-bottom:20px;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/8/8a/Aalto-Helsinki_propane_2_successful_Gibson.png" style="width:210px;"/> | ||
+ | <figcaption>Assembled Propane 2 insert.</figcaption> | ||
+ | </figure> | ||
+ | <li><p>Used NEBuilder (Gibson) Assembly to construct the BioBricks | ||
+ | <ul> | ||
+ | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts#propane_1">Submitted a BioBrick</a> (Propane 1) containing three crucial enzymes of the propane pathway and inserted it in pSB6A1 backbone for usage in propane production</p></li> | ||
+ | <li><p>After trying ELIC x1, OE-PCR x1 and Gibson assembly x2, we managed to assemble Propane 2 with Gibson assembly and detect the right size insert with colony PCR. However, as we ran out of time, we were unable to successfully propagate the correct plasmid, despite multiple tries. As we didn’t manage to assemble the whole pathway, we couldn’t try out whether the pathway was functional. | ||
+ | </p></li> | ||
+ | </ul></p></li> | ||
</ul> | </ul> | ||
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</section> | </section> | ||
<!-- Propane pathway above --> | <!-- Propane pathway above --> | ||
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<!-- Continuous production below --> | <!-- Continuous production below --> | ||
<section id="continuous" data-anchor="continuous"> | <section id="continuous" data-anchor="continuous"> | ||
− | < | + | <div style="clear:both;"></div> |
<figure style="float:right;margin-left:20px;"> | <figure style="float:right;margin-left:20px;"> | ||
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<figcaption>Our chemostat.</figcaption> | <figcaption>Our chemostat.</figcaption> | ||
</figure> | </figure> | ||
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+ | <h2 >Continuous production</h2> | ||
<h3>Background</h3> | <h3>Background</h3> | ||
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<h2>Cellulose degradation</h2> | <h2>Cellulose degradation</h2> | ||
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+ | <figure style="float:right;margin-left:20px;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/3/3e/Aalto-Helsinki_cellulose_successful_OE-PCR.png" style="width:150px;"/> | ||
+ | <figcaption>Cellulose insert.</figcaption> | ||
+ | </figure> | ||
<h3>Background</h3> | <h3>Background</h3> | ||
− | <p>Well over a 100 million tonnes of cellulosic waste is left unused each year in the European Union alone. To elevate the microbially produced propane to a 2nd generation biofuel and avoid interfering with food production, we wanted to incorporate a plasmid for cellulose hydrolysis into the same bacteria that produces the propane. The plasmid contains three genes encoding the enzymes | + | <p>Well over a 100 million tonnes of cellulosic waste is left unused each year in the European Union alone. To elevate the microbially produced propane to a 2nd generation biofuel and avoid interfering with food production, we wanted to incorporate a plasmid for cellulose hydrolysis into the same bacteria that produces the propane. The plasmid contains three genes encoding the enzymes that hydrolyse cellulose polymers into glucose.</p> |
<h3>Outcome</h3> | <h3>Outcome</h3> | ||
<ul style="list-style-type:disc"> | <ul style="list-style-type:disc"> | ||
<li><p>Looked into <a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_cellulose">modeling cellulose breakdown</a>, but found that there was not enough information to model the breakdown sufficiently well to get any practical benefit from the model.</p></li> | <li><p>Looked into <a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_cellulose">modeling cellulose breakdown</a>, but found that there was not enough information to model the breakdown sufficiently well to get any practical benefit from the model.</p></li> | ||
− | <li><p> | + | <li><p>After trying Gibson assembly x2 and ELIC x2, we were able to produce the Cellulose insert containing the genes encoding three enzymes required for cellulose hydrolysis with OE-PCR. However, due to its low concentration, we were unable to transfer it to a backbone and propagate it.</p></li> |
</ul> | </ul> | ||
Revision as of 15:03, 18 September 2015