Difference between revisions of "Team:Aalto-Helsinki/Results"
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<ul id="sidenav" class="nav nav-stacked"><!-- nav-pills if we want rounded corners --> | <ul id="sidenav" class="nav nav-stacked"><!-- nav-pills if we want rounded corners --> | ||
− | <li><a href="#" data-scroll="overview"><h3>Overview</h3></a></li> | + | <li><a href="#" data-scroll="overview"><h3 style="font-size:21px;">Overview</h3></a></li> |
− | <li><a href="#" data-scroll="propanepathway"><h3>Propane pathway</h3></a></li> | + | <li><a href="#" data-scroll="propanepathway"><h3 style="font-size:21px;">Propane pathway</h3></a></li> |
− | <li><a href="#" data-scroll="continuous"><h3>Continuous production</h3></a></li> | + | <li><a href="#" data-scroll="continuous"><h3 style="font-size:21px;">Continuous production</h3></a></li> |
− | <li><a href="#" data-scroll="cellulose"><h3>Cellulose degradation</h3></a></li> | + | <li><a href="#" data-scroll="cellulose"><h3 style="font-size:21px;">Cellulose degradation</h3></a></li> |
− | <li><a href="#" data-scroll="amphiphilic"><h3>Amphiphilic protein</h3></a></li> | + | <li><a href="#" data-scroll="amphiphilic"><h3 style="font-size:21px;">Amphiphilic protein</h3></a></li> |
− | <li><a href="#" data-scroll="gfp"><h3 | + | <li><a href="#" data-scroll="gfp"><h3 style="font-size:21px;">Fusable GFP</h3></a></li> |
− | <li><a href="#" data-scroll="hp"><h3>Combining modeling and experimental work in iGEM</h3></a></li> | + | <li><a href="#" data-scroll="hp"><h3 style="font-size:21px;">Combining modeling and experimental work in iGEM</h3></a></li> |
− | <li><a href="#" data-scroll="software"><h3> | + | <li><a href="#" data-scroll="software"><h3 style="font-size:21px;">Software</h3></a></li> |
− | <li><a href="#"><h3 style="border-top:solid;">To the top</h3></a></li> | + | <li><a href="#" data-scroll="future"><h3 style="font-size:21px;">Future</h3></a></li> |
+ | <li><a href="#"><h3 style="border-top:solid;font-size:21px;" >To the top</h3></a></li> | ||
</ul> | </ul> | ||
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<section id="overview" class="active" data-anchor="overview"> | <section id="overview" class="active" data-anchor="overview"> | ||
− | <h2 >Overview</h2> | + | <h2>Overview</h2> |
− | + | <ul style="list-style-type:disc"> | |
− | + | <li><p>Extensively <a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_propane">modeled</a> a microbial pathway for propane production and used this information to improve experimental design</p></li> | |
+ | <li><p>Studied the idea of using synthetic amphiphilic micelle-forming proteins as molecular scaffolds to place enzymes in close proximity to each other and modeled both <a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_micelle">micelle formation</a> and <a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_synergy">the effect of enzyme proximity</a> on reaction pathways when competing enzymes are present</p></li> | ||
+ | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts">Submitted three BioBricks</a> to the registry: one containing enzymes of the propane pathway, one encoding a N-terminally fusable GFP and one unlikely containing the amphiphilic protein</p></li> | ||
+ | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Design">First report ever</a> of successful continuous microbial propane production</p></li> | ||
+ | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Questionnaire">Studied</a> the relationship of modeling and experimentation in iGEM teams, as well as the educational background of iGEM participants</p></li> | ||
+ | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Software">Built</a> and <a href="https://2015.igem.org/Team:Aalto-Helsinki/humhub">ideated</a> software to help iGEM teams better collaborate and communicate</p></li> | ||
+ | </ul> | ||
</p> | </p> | ||
<!-- Overview above --> | <!-- Overview above --> | ||
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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. | + | <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. Our 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">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 <i>E. coli</i> |
− | + | <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 both ELIC and OE-PCR once and Gibson assembly twice, 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> | ||
− | |||
</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> | ||
+ | |||
+ | <h2 >Continuous production</h2> | ||
<h3>Background</h3> | <h3>Background</h3> | ||
− | <p>Industrial biopropane production would most likely occur as a continuously operated process, as it is economically more feasible in large scale production. | + | <p>Industrial biopropane production would most likely occur as a continuously operated process, as it is economically more feasible in large scale production. To take the first step towards industrial scale production we wanted to try continuous production of propane using a <i>E. coli</i> strain provided to us by Pauli Kallio from the University of Turku.</p> |
<h3>Outcome</h3> | <h3>Outcome</h3> | ||
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<li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Design">First report ever</a> worldwide of successful microbial production of propane in continuous production</p></li> | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Design">First report ever</a> worldwide of successful microbial production of propane in continuous production</p></li> | ||
<li><p>145 hour continuous production experiment in a 500 ml chemostat</p></li> | <li><p>145 hour continuous production experiment in a 500 ml chemostat</p></li> | ||
− | <li><p>Propane yield 22 | + | <li><p>Propane yield up to 22.7 µg/L in reactor gas phase</p></li> |
</ul> | </ul> | ||
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<h2>Cellulose degradation</h2> | <h2>Cellulose degradation</h2> | ||
+ | |||
+ | <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 | + | <p>Well over 200 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 and ELIC twice, 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> | ||
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<h3>Background</h3> | <h3>Background</h3> | ||
− | <p>Amphiphilic proteins are synthetic proteins consisting of a hydrophilic and a hydrophobic domain that have been shown to spontaneously form micellar and vesicular structures. We were interested whether these structures could be used as scaffolds to have subsequent enzymes of the propane pathway in close proximity. | + | <p>Amphiphilic proteins are synthetic proteins consisting of a hydrophilic and a hydrophobic domain that have been shown to spontaneously form micellar and vesicular structures. We were interested whether these structures could be used as scaffolds to have subsequent enzymes of the propane pathway in close proximity. We wanted to model whether fusing enzymes to the proteins would disrupt micelle formation and whether our idea could enhance propane output.</p> |
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<ul style="list-style-type:disc"> | <ul style="list-style-type:disc"> | ||
− | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_synergy">Built a stochastic synergy model</a> in Python, simulating enzyme function in cases where two subsequent enzymes stay in close proximity to each other as opposed to moving around | + | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_synergy">Built a stochastic synergy model</a> in Python, simulating enzyme function in cases where two subsequent enzymes stay in close proximity to each other as opposed to moving around independently in a cell</p></li> |
<li><p>The synergy model predicts a 200-400 % increase in product output if enzymes stay in close proximity to each other</p></li> | <li><p>The synergy model predicts a 200-400 % increase in product output if enzymes stay in close proximity to each other</p></li> | ||
<li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_micelle">Constructed a geometrical micelle model</a> based on the sizes and structures of the micelle-forming proteins, indicating that it is indeed possible for micellar structures to form even as enzymes are fused to them</p></li> | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Modeling_micelle">Constructed a geometrical micelle model</a> based on the sizes and structures of the micelle-forming proteins, indicating that it is indeed possible for micellar structures to form even as enzymes are fused to them</p></li> | ||
− | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts">Submitted a BioBrick</a> encoding the amphiphilic protein to the registry</p></li> | + | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts#amphiphilic">Submitted a BioBrick</a> unlikely encoding the amphiphilic protein to the registry</p></li> |
<li><p>Due to time restraints, we were unable to experimentally validate the idea by fusing either two subsequent enzymes of the propane pathway or components of the violacein pathway to the amphiphilic proteins</p></li> | <li><p>Due to time restraints, we were unable to experimentally validate the idea by fusing either two subsequent enzymes of the propane pathway or components of the violacein pathway to the amphiphilic proteins</p></li> | ||
</ul> | </ul> | ||
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<h3>Background</h3> | <h3>Background</h3> | ||
− | <p>To validate our amphiphilic brick, we needed a GFP that could be fused to the amino-terminal end of the protein. There was no such brick | + | <p>To validate our amphiphilic brick, we needed a GFP that could be fused to the amino-terminal end of the protein. There was no such brick in the distribution kit. We also thought there was no such brick in the registry, but later came to realize we were wrong. We wanted to create a GFP BioBrick that could be fused to the N-terminal end of any protein using BioBrick methods.</p> |
<h3>Outcome</h3> | <h3>Outcome</h3> | ||
<ul style="list-style-type:disc"> | <ul style="list-style-type:disc"> | ||
− | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts">Submitted a BioBrick</a> encoding GFP with an extra nucleotide prior to the suffix, ensuring that it can be fused to the N-terminal end of a protein using BioBrick assembly methods while maintaining the reading frame</p></li> | + | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts#gfp">Submitted a BioBrick</a> encoding GFP with an extra nucleotide prior to the suffix, ensuring that it can be fused to the N-terminal end of a protein using BioBrick enzyme assembly methods while maintaining the reading frame</p></li> |
<li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Collaborations">Collaborated</a> with team HS Slovenia to validate the brick</p></li> | <li><p><a href="https://2015.igem.org/Team:Aalto-Helsinki/Collaborations">Collaborated</a> with team HS Slovenia to validate the brick</p></li> | ||
</ul> | </ul> | ||
<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/2015/8/82/Aalto-Helsinki_GFP-amph_vs_ctrl.jpg" style="width:100%;"/> | + | <img src="https://static.igem.org/mediawiki/2015/archive/8/82/20150918182105!Aalto-Helsinki_GFP-amph_vs_ctrl.jpg" style="width:100%;"/> |
<figcaption><b>Figure 3:</b> Upper row: <i>E. coli</i> expressing GFP fused to the N-terminal end of our amphiphilic protein. Bottom row: control. On the left, a light microscope picture and in the middle a fluorescent microscope picture of the same cells (excitation at 488 nm, detection 493-598 nm). On the right the two pictures to the left merged, showing that GFP is expressed in transformed cells, but not in control cells.</figcaption> | <figcaption><b>Figure 3:</b> Upper row: <i>E. coli</i> expressing GFP fused to the N-terminal end of our amphiphilic protein. Bottom row: control. On the left, a light microscope picture and in the middle a fluorescent microscope picture of the same cells (excitation at 488 nm, detection 493-598 nm). On the right the two pictures to the left merged, showing that GFP is expressed in transformed cells, but not in control cells.</figcaption> | ||
</figure> | </figure> | ||
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<li><p>Biggest issues in collaboration between modelers and experimentalists are: lack of knowledge of the other field, lack of common terminology and differences in ways of thinking</p></li> | <li><p>Biggest issues in collaboration between modelers and experimentalists are: lack of knowledge of the other field, lack of common terminology and differences in ways of thinking</p></li> | ||
<li><p>Both modelers and biologists need to understand the basics of the other field to be able to effectively collaborate.</p></li> | <li><p>Both modelers and biologists need to understand the basics of the other field to be able to effectively collaborate.</p></li> | ||
− | <li><p>Having experimentalists and modelers work close together is beneficial. One approach generally found successful is to have some biologists get involved in modeling to help ensure models are useful for the project and connected to reality.</p></li> | + | <li><p>Having experimentalists and modelers work close together is beneficial. One approach generally found successful is to have some biologists get involved in modeling to help ensure models are useful for the project and connected to reality. Modelers can too take some time to get familiar with the wetlab in the beginning of the project</p></li> |
<li><p>Regular team meetings for presenting and discussing progress and issues of every field take time, but ensure all team members stay informed and can voice their insights.</p></li> | <li><p>Regular team meetings for presenting and discussing progress and issues of every field take time, but ensure all team members stay informed and can voice their insights.</p></li> | ||
<li><p>Students with a mathematical background are underrepresented in iGEM teams as compared to professional synthetic biology groups. </p></li> | <li><p>Students with a mathematical background are underrepresented in iGEM teams as compared to professional synthetic biology groups. </p></li> | ||
− | <li><p> | + | <li><p>iGEM teams have relatively many biotechnology students, who often stand seem to act as mediators between the modeling and experimentation.</p></li> |
</ul> | </ul> | ||
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<section id="software" data-anchor="software"> | <section id="software" data-anchor="software"> | ||
− | <h2>HumHub report and Collab Seeker</h2> | + | <h2>Software: HumHub report and Collab Seeker</h2> |
<h3>Background</h3> | <h3>Background</h3> | ||
− | <p>Finding new collaboration partners in iGEM is not easy, as finding information about different teams projects is time-consuming and often difficult. On the other hand, communication with other teams as well as internal team communication can be difficult due to a multitude of platforms used, often cluttered with non-iGEM content. We wanted to do something to these issues to make iGEM even better.</p> | + | <p>Finding new collaboration partners in iGEM is not easy, as finding information about different teams' projects is time-consuming and often difficult. On the other hand, communication with other teams as well as internal team communication can be difficult due to a multitude of platforms used, often cluttered with non-iGEM content. We wanted to do something to these issues to make iGEM even better.</p> |
<h3>Outcome</h3> | <h3>Outcome</h3> | ||
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<!-- HumHub report and Collab Seeker above --> | <!-- HumHub report and Collab Seeker above --> | ||
− | + | <section id="future" data-anchor="future"> | |
− | < | + | <h2>Future</h2> |
<p>To read our thoughts on future prospects and on how to carry on from where we left, please see our <a href="https://2015.igem.org/Team:Aalto-Helsinki/Future">Future page</a>.</p> | <p>To read our thoughts on future prospects and on how to carry on from where we left, please see our <a href="https://2015.igem.org/Team:Aalto-Helsinki/Future">Future page</a>.</p> | ||
+ | <h2>Submitted BioBricks and Achievements</h2> | ||
− | + | <p>To read about the BioBricks we submitted, see our <a href="https://2015.igem.org/Team:Aalto-Helsinki/Parts">Submitted Parts page</a>. To see how our achievements match with the medal criteria, see our <a href="https://2015.igem.org/Team:Aalto-Helsinki/Medals">Checklist</a>.</p> | |
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Latest revision as of 04:31, 29 October 2015