http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=MaggieRY&year=&month=2012.igem.org - User contributions [en]2024-03-29T11:55:47ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:53:33Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Post-Regionals">Post-Regionals</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
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TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
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<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
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<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/tkwafeHnG-U" frameborder="0" allowfullscreen></iframe><br />
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<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
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<br />
<p>In short, the bioreactor was filled with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be used for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing an <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:52:17Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|proj_hp|<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
<br />
<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be used for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing an <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:51:31Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
<br />
<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be used for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing a <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:49:25Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
<br />
<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be turn on this device to allow for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing a <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:48:33Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|proj_hp|<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
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<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
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<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
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<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
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<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
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<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
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<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
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<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
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<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
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<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
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<h2> Taking FRED out to the field! </h2><br />
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<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
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<p> We also created a video to show how we would test this water sample with our prototype and software package. This video can be found below.</p><br />
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<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
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</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
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<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
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<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be turn on this device to allow for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
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</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
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<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing a <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
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<br><h2>Why Choose Hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
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<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of disrupting metabolism or the limitations of a single channel output.</p><br />
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<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
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[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
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<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
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<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
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<h2>Genes, Chemicals, and Circuits</h2><br />
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<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
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[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
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<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
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[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
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<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
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<h2>Does it Work?</h2><br />
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<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
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[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
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<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
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<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
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<h2>What Next?</h2><br />
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<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
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</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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</html><br />
[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
<br />
<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
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</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
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</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
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<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
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<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
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<h3> Results </h3><br />
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<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
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<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
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<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
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<h3> Kill Gene Testing </h3><br />
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<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
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<h3> Results </h3><br />
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</html>[[File:24 hour assay with mgtAp-mgtArb-S7 Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
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<h2>An alternative: a glucose repressible system</h2><br />
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<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
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</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T02:25:29Z<p>MaggieRY: </p>
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TITLE=Regulation/Expression Platform|<br />
CONTENT=<br />
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<img src="https://static.igem.org/mediawiki/2012/8/8c/UCalgary2012_FRED_Killswitch_Regulation_Low-Res.png" style="float: right; padding: 10px; height: 200px;"></img><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
<br />
</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
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<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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</html><br />
[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
<br />
</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<br />
<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
<br />
</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
<br />
<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
<br />
<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
<br />
<h3> Results </h3><br />
<br />
<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
<br />
<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
<br />
<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
<br />
<h3> Kill Gene Testing </h3><br />
<br />
<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
<br />
<h3> Results </h3><br />
<br />
</html>[[File:24 hour assay with mgtAp-mgtArb-S7 Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
<br />
<br />
<h2>An alternative: a glucose repressible system</h2><br />
<br />
<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
<html><br />
<br />
</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
<html><br />
<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T02:20:52Z<p>MaggieRY: </p>
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TITLE=Regulation/Expression Platform|<br />
CONTENT=<br />
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<img src="https://static.igem.org/mediawiki/2012/8/8c/UCalgary2012_FRED_Killswitch_Regulation_Low-Res.png" style="float: right; padding: 10px; height: 200px;"></img><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
<br />
</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
<br />
<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
<br />
<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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</html><br />
[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
<br />
<h2> The Moco Riboswitch </h2><br />
<br />
<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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<br />
</html><br />
[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
<br />
</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<br />
<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
<br />
<h3> GFP testing</h3><br />
<br />
</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
<br />
<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
<br />
<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
<br />
<h3> Results </h3><br />
<br />
<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
<br />
<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
<br />
<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
<br />
<h3> Kill Gene Testing </h3><br />
<br />
<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
<br />
<h3> Results </h3><br />
<br />
</html>[[File:24 hour assay with <i>mgtAp-mgtArb-S7</i> Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
<br />
<br />
<h2>An alternative: a glucose repressible system</h2><br />
<br />
<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T01:40:38Z<p>MaggieRY: </p>
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
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</html> [[File:UofC_RIBOSWITCH.png|centre|350px]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 1: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
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<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 2: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
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<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 3. </p><br />
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</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 3: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
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</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 4: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
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<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 4 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
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<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
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<h3> Results </h3><br />
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<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 5. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 5: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
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<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
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<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
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<h3> Kill Gene Testing </h3><br />
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<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
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<h3> Results </h3><br />
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</html>[[File:24 hour assay with mgtap-rb-S7 Ucalgary.png|thumb|750px|center| Figure 6: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 6 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
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<h2>An alternative: a glucose repressible system</h2><br />
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<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 7). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 7: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 8: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our flux variability analysis showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our Synergy Page for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T01:08:43Z<p>MaggieRY: </p>
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
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</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
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<h2>Incorporating human practices in the design of our system </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’ve made on our project with us. We got a variety of different perspectives from suggestions on the...... The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, we have OSCAR components, and we have some killswitch components, but how functional are some of these parts, and how do they work together. So our next major goal was to <b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d actually gotten.</p><br />
<br />
<h2> Putting FRED together </h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given hat</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<h2> Putting together our killswitches </h2><br />
<br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. So we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2> Putting our Killswitch into OSCAR</h2><br />
<br />
<p>The next thing we wanted to validate was that our glycine knockout strain would in fact work as we wanted it to in OSCAR. Namely, we wanted to know if putting the PetroBrick into our glycine knockout strain and growing it in the presence of glycine would still give us the same increased hydrocarbon production that we saw when validating our model. We transformed the PetroBrick into the knockout strain and repeated the PetroBrick validation assay protocol. Our results are shown below:</p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to to perform a kind of field test with FRED, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be absolutely assured that it won’t be released. However, we could test tailings water with our biosensor prototype in the lab. Here is the data for this test. </p><br />
<br />
<H2> Putting OSCAR in action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached Kelly Roberge, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (1000L+ tanks). To start with, we would need to consider the amount of naphthenic acids needed to provide steady throughput in our system and just how much hydrocarbon can be produced in a full cycle of our system. He recommended that we use computer modelling to explore these challenges. This could allow us to determine the possible hydrocarbon output of our lab scale experiments once they are up and running. Additionally, we would need to take into consideration the composition of tailings pond solution, especially the sludge and bitumen content. The sludge could be physically harmful to our bioreactor and reduce its overall efficiency as well. A possible way to tackle this challenge would be to use current mature fine tailings drying techniques used to help speed the reuse of water in the tailings ponds. As tailings fines settle the resulting tailings water component would be left behind. This would be an ideal input into our system for potential remediation and production of hydrocarbons as it would contain a large proportion of the compounds thought to be most toxic in the tailings. By using this matured tailings as the input to our system it could help increase the efficiency of our bioreactor and provide for a smoother scale up from the lab bench to an industrial bioreactor.</p><br />
<br />
<h2>Glycine Auxotrophy Still Allows For Hydrocarbon Production</h2><br />
<p><b>In fact its better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure X: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure X: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure X: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-26T23:49:39Z<p>MaggieRY: </p>
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<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the industry where it would be employed. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
</p><br />
<br />
<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
<p>We spoke with Christine Daly, an Aquatic Reclamation Research Coordinator at Suncor Energy Inc. Christine expressed an interest in our <a href="https://2011.igem.org/Team:Calgary">project in 2011</a> and was willing to discuss this year’s project design with us. One major point that was brought up early on in our design was that there is an opportunity for engineered organisms to outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar concern. To address these concerns, we created our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> system, which would physically contain our bacteria, and also a <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">genetic killswitch mechanism</a>. Another interesting point brought up in this discussion was how the oil industry is currently looking into biology as one of many potential alternative methods to remediate the toxic components of tailings ponds and the oil sands in general. Research exists with other systems such as algal bioremediation, but practical implementations of biology in the oil sands appear to be rather few and far between. Oil industries do, however, appear to show an increased interest in biology (and in turn, synthetic biology) as a possible solution to various problems, a sentiment reflected in <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">our dialogue with the Oil Sands Leadership Initiative</a>.</p><br />
<p>The full interview can be viewed below.</p><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/GiM6EIC9XBo" frameborder="0" allowfullscreen></iframe><br />
<br />
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<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/86XQ-Kg5fJ4" frameborder="0" allowfullscreen></iframe><br />
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<br />
<a name="postregionals"></a><br />
<h2>Follow-Up Interviews</h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
<br />
<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
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<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-26T20:43:03Z<p>MaggieRY: </p>
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
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TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<br />
<h2>Incorporating human practices in the design of our system </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. In order to ensure we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’ve made on our project with us. We got a variety of different perspectives from suggestions on the...... The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, we have OSCAR components, and we have some killswitch components, but how functional are some of these parts, and how do they work together. So our next major goal was to <b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d actually gotten.</p><br />
<br />
<h2> Putting FRED together </h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given hat</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<h2> Putting together our killswitches </h2><br />
<br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. So we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2> Putting our Killswitch into OSCAR</h2><br />
<br />
<p>The next thing we wanted to validate was that our glycine knockout strain would in fact work as we wanted it to in OSCAR. Namely, we wanted to know if putting the PetroBrick into our glycine knockout strain and growing it in the presence of glycine would still give us the same increased hydrocarbon production that we saw when validating our model. We transformed the PetroBrick into the knockout strain and repeated the PetroBrick validation assay protocol. Our results are shown below:</p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that FRED could actually detect toxins found in tailings ponds within a laboratory setting, the next challenge would be to take FRED out to a tailings pond to out him to the test. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. We still felt that testing FRED out in the real world, demonstrating that our prototype was easy to use and functional outside of a lab environment was extremely important. As such, we decided to do a field test of a body of water in our own city. The first thing we worried about though, was if there was any regulation surrounding water sampling, or performing a field test with a genetically modified organism (GMO). So we did a literature search to look for any regulations that might exist. We couldn’t find anything that pertained to our province, so we looked to Ontario and the United States. We looked at the concise guide to U.S. federal guidelines, rules and regulations for synthetic biology. In this guide, rules pertaining to field tests are covered. In cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification.</p><br />
<br />
<H2> Putting OSCAR in action! </h2><br />
<p> Once we had tested FRED and showed that he is not only able to , we wanted to put OSCAR into action and who that the design of our bioreactor was capable of doing what we wanted it to. By the end of the summer, we had a lab scale prototype and design for our bioreactor. To help us move forward with this portion of the project we interviewed Kelly Roberge, a consultant for oil sands specializing in tailings ponds. This interview gave us much to think about and helped us form ideas on how to improve the overall design of our bioreactor. In particular, Kelly’s advice and questions surrounded scaling up our bioreactor to industrial size. <br />
There are many things to consider when going from lab scale to industrial scale, and very little can be correlated linearly when moving from lab scale to industrial size (1000L+ tanks). To start, we would have to consider the amount of naphthenic acids needed to provide steady throughput in our system, and how much hydrocarbon can be produced in a full cycle of our system. To help provide theoretical solutions to these issues, we could determine the hydrocarbon output of our lab scale experiments once they are up and running to get an idea of what kind of numbers we are dealing with.<br />
In addition, we will have to take into consideration the composition of tailings pond solution, especially the sludge and bitumen content. This sludge could be harmful to our bioreactor and reduce the efficiency as well. One way we could solve this problem is by utilizing current NFT drying techniques used to help degrade tailings ponds. A sludge reduced water runoff is the result of this process. This water runoff could be the input to our system, which still contains large quantities of naphthenic acids. This would help increase the efficiency of our bioreactor and even allow scale up to be possible. </p><br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
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<h2>Incorporating human practices in the design of our system </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. In order to ensure we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’ve made on our project with us. We got a variety of different perspectives from suggestions on the...... The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, we have OSCAR components, and we have some killswitch components, but how functional are some of these parts, and how do they work together. So our next major goal was to <b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d actually gotten.</p><br />
<br />
<h2> Putting FRED together </h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given hat</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<h2> Putting together our killswitches </h2><br />
<br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. So we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2> Putting our Killswitch into OSCAR</h2><br />
<br />
<p>The next thing we wanted to validate was that our glycine knockout strain would in fact work as we Wanted it to in OSCAR. Namely, we wanted to know if putting the PetroBrick into our glycine knockout strain and growing it in the presence of glycine would still give us the same increased hydrocarbon production that we saw when validating our model. We transformed the PetroBrick into the knockout strain and repeated the PetroBrick validation assay protocol. Our results are shown below:</p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that FRED could actually detect toxins found in tailings ponds within a laboratory setting, the next challenge would be to take FRED out to a tailings pond to out him to the test. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. We still felt that testing FRED out in the real world, demonstrating that our prototype was easy to use and functional outside of a lab environment was extremely important. As such, we decided to do a field test in a body of water in our own city. The first thing we worried about though, was if there was any regulation surrounding water sampling, or performing a field test with a genetically modified organism (GMO). So we did a literature search to look for any regulations that might exist. We couldn’t find anything that pertained to our province, so we looked to Ontario and the United States. We looked at the concise guide to U.S. federal guidelines, rules and regulations for synthetic biology. In this guide, rules pertaining to field tests are covered. In cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification.</p><br />
<br />
<H2> Putting OSCAR in action! </h2><br />
<p> Once we had tested FRED and showed that he is not only able to , we wanted to put OSCAR into action and who that the design of our bioreactor was capable of doing what we wanted it to. By the end of the summer, we had a lab scale prototype and design for our bioreactor. To help us move forward with this portion of the project we interviewed Kelly Roberge, a consultant for oil sands specializing in tailings ponds. This interview gave us much to think about and helped us form ideas on how to improve the overall design of our bioreactor. In particular, Kelly’s advice and questions surrounded scaling up our bioreactor to industrial size. <br />
There are many things to consider when going from lab scale to industrial scale, and very little can be correlated linearly when moving from lab scale to industrial size (1000L+ tanks). To start, we would have to consider the amount of naphthenic acids needed to provide steady throughput in our system, and how much hydrocarbon can be produced in a full cycle of our system. To help provide theoretical solutions to these issues, we could determine the hydrocarbon output of our lab scale experiments once they are up and running to get an idea of what kind of numbers we are dealing with.<br />
In addition, we will have to take into consideration the composition of tailings pond solution, especially the sludge and bitumen content. This sludge could be harmful to our bioreactor and reduce the efficiency as well. One way we could solve this problem is by utilizing current NFT drying techniques used to help degrade tailings ponds. A sludge reduced water runoff is the result of this process. This water runoff could be the input to our system, which still contains large quantities of naphthenic acids. This would help increase the efficiency of our bioreactor and even allow scale up to be possible. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-26T09:53:55Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similarly sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|Figure 1: Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the bioreactors. ]]<html><br />
<p>Before diving into making a bioreactor, we first had to research current solutions in the field. To help us with this phase, we read papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to be considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors doing research on bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Here is a picture from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable was a cross between a fed-batch system and a continuous stir method in a closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. To remove the hydrocarbons from the culture we decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons, which is favored for an industrial scale (1000+ L tanks). Tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p> <br />
<br />
<p>To make sure that the belt does not transfer live bacteria into the hydrocarbon collection tank, we will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of radiation just before the hydrocarbons are removed from the bioreactor chamber.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|Figure 2: From computer to prototype: how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took shape using materials we found in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is the first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. Since our bioreactor would necessarily have live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
<br />
<h2>The Belt Skimmer in Action</h2><br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video showcase our belt skimmer. The model hydrocarbons stick to the belt shown in the video and are skimmed off into a Tim Horton's coffee cup (the only disposable cup we could find in the lab. </p><br />
<br />
<h2>Current Prototype with both the Sparger and Turbine</h2><br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> The second video shows our bioreactor prototype in action with both the sparger and turbine running. </p><br />
<br />
<h2>Belt Skimmer with a Collection Chamber</h2><br />
<div align="center"><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/HtcgPG3reH4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video shows the collection chamber we added to the bioreactor. There is a hole at the bottom of the chamber so that during the bioreactors' showcase the model hydrocarbons don't accumulate in the chamber. </p><br />
<br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using the Maya and RealFlow programs. Maya is a complex and extremely versatile computer animation program used in many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
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<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
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<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
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<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
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<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|Figure 3: Left panel: Belt rankings from three different tests. We tested the ability of the belts to pick up hydrocarbons, and to exclude bacteria or tailings pond water. Right panel: The five belts we tested and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution so as to prevent the settling of bacterial cells and other heavier materials and to ensure even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for aerobic bacteria to thrive. When assembled together, the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger, a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. In order to use the air sparger, we decided to use a HEPA filter to screen air coming out of the system to maintain constant pressure in the tank. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|Figure 4: This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was a very encouraging result since we want to skim the hydrocarbon products from the top layer of our bioreactor.]]<html><br />
<br />
<p> Furthermore, we tested our belts' ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, many different types of NA's were represented in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This data suggests that minimal NA's were found in our skimmed hydrocarbon layer and that most were left in the water layer. <br />
</html>[[File:HC layer, skimmed (no NaOH)-ucalgary.png|thumb|600px|center|Figure 5: This graph shows the amount of NA's found in the skimmed hydrocarbon layer. Each bar represents the carbon ring count for a different type of NA. Clearly, minimal NA's were skimmed into the hydrocarbon layer.]]<html><br />
</html>[[File:Water layer, skimmed-ucalgary.png|thumb|600px|centre|Figure 6: This graph shows how many NA's were left in the water layer of our skimmed solution. This data suggests that minimal amounts of NA were skimmed into our solution, and with most of those skimmed found in the water layer.]] <html><br />
<br />
<br />
<h2>The Final System</h2><br />
<p>Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR bacterium would have the most suitable kill-switch circuit attached to its respective hydrocarbon conversion circuit. The bacterium would also have a deletion of a gene for the biosynthesis of glycine. Glycine would be supplemented in our defined production media, but cells would not survive outside of the bioreactor where glycine is absent. We envision OSCAR to be a co-culture of decarboxylation, decatecholization, denitrogenation, and desulfurization. Lastly, due to the energetically expensive nature of maintaining the circuits, we anticipate that if the circuits are constitutively produced cell growth rate may be very slow. Therefore in the final circuits we may want them to be activated by quorum sensing promoter systems. Essentially, when cells are at low density they focus energy on growth; when cells reach appropriate density for the reaction chamber, transcription of the circuit is enabled. Together we hope that the system will clean up recalcitrant petroleum waste and produce energy.<br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-26T09:49:47Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
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<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similarly sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|Figure 1: Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the bioreactors. ]]<html><br />
<p>Before diving into making a bioreactor, we first had to research current solutions in the field. To help us with this phase, we read papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to be considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors doing research on bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Here is a picture from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable was a cross between a fed-batch system and a continuous stir method in a closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. To remove the hydrocarbons from the culture we decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons, which is favored for an industrial scale (1000+ L tanks). Tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p> <br />
<br />
<p>To make sure that the belt does not transfer live bacteria into the hydrocarbon collection tank, we will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of radiation just before the hydrocarbons are removed from the bioreactor chamber.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|Figure 2: From computer to prototype: how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took shape using materials we found in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is the first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. Since our bioreactor would necessarily have live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
<br />
<h2>The Belt Skimmer in Action</h2><br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video showcase our belt skimmer. The model hydrocarbons stick to the belt shown in the video and are skimmed off into a Tim Horton's coffee cup (the only disposable cup we could find in the lab. </p><br />
<br />
<h2>Current Prototype with both the Sparger and Turbine</h2><br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> The second video shows our bioreactor prototype in action with both the sparger and turbine running. </p><br />
<br />
<h2>Belt Skimmer with a Collection Chamber</h2><br />
<div align="center"><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/HtcgPG3reH4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video shows the collection chamber we added to the bioreactor. There is a hole at the bottom of the chamber so that during the bioreactors' showcase the model hydrocarbons don't accumulate in the chamber. </p><br />
<br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using the Maya and RealFlow programs. Maya is a complex and extremely versatile computer animation program used in many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
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<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
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<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
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<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|Figure 3: Left panel: Belt rankings from three different tests. We tested the ability of the belts to pick up hydrocarbons, and to exclude bacteria or tailings pond water. Right panel: The five belts we tested and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution so as to prevent the settling of bacterial cells and other heavier materials and to ensure even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for aerobic bacteria to thrive. When assembled together, the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger, a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. In order to use the air sparger, we decided to use a HEPA filter to screen air coming out of the system to maintain constant pressure in the tank. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|Figure 4: This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was a very encouraging result since we want to skim the hydrocarbon products from the top layer of our bioreactor.]]<html><br />
<br />
<p> Furthermore, we tested our belts' ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, many different types of NA's were represented in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This data suggests that minimal NA's were found in our skimmed hydrocarbon layer and that most were left in the water layer. <br />
</html>[[File:HC layer, skimmed (no NaOH)-ucalgary.png|thumb|600px|center|Figure 5: This graph shows the amount of NA's found in the skimmed hydrocarbon layer. Each bar represents the carbon ring count for a different type of NA. Clearly, minimal NA's were skimmed into the hydrocarbon layer.]]<html><br />
</html>[[File:Water layer, skimmed-ucalgary.png|thumb|600px|centre|Figure 6: This graph shows how many NA's were left in the water layer of our skimmed solution. This data suggests that minimal amounts of NA were skimmed into our solution, and with most of those skimmed found in the water layer.]] <html><br />
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<br />
<h2>The Final System</h2><br />
<p>Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR bacterium would have the most suitable kill-switch circuit attached to its respective hydrocarbon conversion circuit. The bacterium would also have a deletion of a gene for the biosynthesis of glycine, which would be supplemented in our defined production media, but would prohibit survival outside of the bioreactor. We envision OSCAR to be a co-culture of decarboxylation, decatecholization, denitrogenation, and desulfurization. Lastly, due to the energetically expensive nature of maintaining the circuits, we anticipate that if the circuits are constitutively produced cell growth rate may be very slow. Therefore in the final circuits we may want them to be activated by quorum sensing promoter systems. Essentially, when cells are at low density they focus energy on growth; when cells reach appropriate density for the reaction chamber, transcription of the circuit is enabled. Together we hope that the system will clean up recalcitrant petroleum waste and produce energy.<br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-26T09:46:50Z<p>MaggieRY: </p>
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<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similarly sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
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<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|Figure 1: Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the bioreactors. ]]<html><br />
<p>Before diving into making a bioreactor, we first had to research current solutions in the field. To help us with this phase, we read papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to be considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors doing research on bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Here is a picture from our trip to the wastewater plant!</p><br />
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<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable was a cross between a fed-batch system and a continuous stir method in a closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. To remove the hydrocarbons from the culture we decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons, which is favored for an industrial scale (1000+ L tanks). Tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p> <br />
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<p>To make sure that the belt does not transfer live bacteria into the hydrocarbon collection tank, we will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of radiation just before the hydrocarbons are removed from the bioreactor chamber.</p><br />
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</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|Figure 2: From computer to prototype: how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took shape using materials we found in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is the first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
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<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. Since our bioreactor would necessarily have live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
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<h2>The Belt Skimmer in Action</h2><br />
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<p> This video showcase our belt skimmer. The model hydrocarbons stick to the belt shown in the video and are skimmed off into a Tim Horton's coffee cup (the only disposable cup we could find in the lab. </p><br />
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<h2>Current Prototype with both the Sparger and Turbine</h2><br />
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<p> The second video shows our bioreactor prototype in action with both the sparger and turbine running. </p><br />
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<h2>Belt Skimmer with a Collection Chamber</h2><br />
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<p> This video shows the collection chamber we added to the bioreactor. There is a hole at the bottom of the chamber so that during the bioreactors' showcase the model hydrocarbons don't accumulate in the chamber. </p><br />
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<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using the Maya and RealFlow programs. Maya is a complex and extremely versatile computer animation program used in many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
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<h2>Particle Simulation Using RealFlow2012</h2><br />
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<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
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<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
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<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|Figure 3: Left panel: Belt rankings from three different tests. We tested the ability of the belts to pick up hydrocarbons, and to exclude bacteria or tailings pond water. Right panel: The five belts we tested and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution so as to prevent the settling of bacterial cells and other heavier materials and to ensure even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for aerobic bacteria to thrive. When assembled together, the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger, a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. In order to use the air sparger, we decided to use a HEPA filter to screen air coming out of the system to maintain constant pressure in the tank. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|Figure 4: This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was a very encouraging result since we want to skim the hydrocarbon products from the top layer of our bioreactor.]]<html><br />
<br />
<p> Furthermore, we tested our belts' ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, many different types of NA's were represented in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This data suggests that minimal NA's were found in our skimmed hydrocarbon layer and that most were left in the water layer. <br />
</html>[[File:HC layer, skimmed (no NaOH)-ucalgary.png|thumb|600px|center|Figure 5: This graph shows the amount of NA's found in the skimmed hydrocarbon layer. Each bar represents the carbon ring count for a different type of NA. Clearly, minimal NA's were skimmed into the hydrocarbon layer.]]<html><br />
</html>[[File:Water layer, skimmed-ucalgary.png|thumb|600px|centre|Figure 6: This graph shows how many NA's were left in the water layer of our skimmed solution. This data suggests that minimal amounts of NA were skimmed into our solution, and with most of those skimmed found in the water layer.]] <html><br />
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<br />
<h2>The Final System</h2><br />
<p>Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR bacterium would have the most suitable kill-switch circuit attached to its respective hydrocarbon conversion circuit. The bacterium would also have a deletion of a gene for the biosynthesis of glycine, which would be supplemented in our defined production media. We envision OSCAR to be a co-culture of decarboxylation, decatecholization, denitrogenation, and desulfurization. Lastly, due to the energetically expensive nature of maintaining the circuits, we anticipate that if the circuits are constitutively produced cell growth rate may be very slow. Therefore in the final circuits we may want them to be activated by quorum sensing promoter systems. Essentially, when cells are at low density they focus energy on growth; when cells reach appropriate density for the reaction chamber, transcription of the circuit is enabled. Together we hope that the system will clean up recalcitrant petroleum waste and produce energy.<br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-26T09:30:24Z<p>MaggieRY: </p>
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<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the industry where it would be employed. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
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<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
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<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
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<h2>Follow-Up Interviews</h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
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<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-26T09:24:17Z<p>MaggieRY: </p>
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<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
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<br><h2>Why choose hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
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<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of disrupting metabolism or the limitations of a single channel output.</p><br />
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<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
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[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
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<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
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<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
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<h2>Genes, Chemicals, and Circuits</h2><br />
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<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
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[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
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<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
<br />
<h2>Does it Work?</h2><br />
<br />
<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
<br />
</html><br />
[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
<html><br />
<br />
<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
<br />
<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
<br />
<h2>What Next?</h2><br />
<br />
<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
<br />
</html>}}}<br />
<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-26T09:03:12Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Novel Electrochemical Reporting System|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_FRED_Reporting_Low-Res.png" style="padding: 10px; width: 225; float: right;"></img><br />
<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
<br />
<br><h2>Why choose hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
<br />
<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of cutting off metabolism or having to use only a single channel output.</p><br />
<br />
<br />
<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
<br />
</html><br />
[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
<html><br />
<br />
<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
<br />
<br><br />
<br />
<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
<br />
<br><br />
<br />
<h2>Genes, Chemicals, and Circuits</h2><br />
<br />
<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
<br />
</html><br />
[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
<html><br />
<br />
<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
<br />
<h2>Does it Work?</h2><br />
<br />
<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
<br />
</html><br />
[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
<html><br />
<br />
<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
<br />
<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
<br />
<h2>What Next?</h2><br />
<br />
<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
<br />
</html>}}}<br />
<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-26T07:38:45Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Novel Electrochemical Reporting System|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_FRED_Reporting_Low-Res.png" style="padding: 10px; width: 225; float: right;"></img><br />
<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
<br />
<br><h2>What's the big idea?</h2><br />
<p>To get our bacteria to report on the toxic compounds present in the tailings ponds we needed a quick and reliable system that would function in a wide variety of environments, including murky solutions. We turned to electrochemistry for this, as the turbidity of the solution doesn't effect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product or to transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are through the action of metal respiration, phosphatases, or hydrolases. </p><br />
<br />
<p>The first system, that of the respiration of metals, involves using an organism that uses ions such as Fe<sup>3+</sup> as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it would require throttling the entire metabolic network of an organism such that it could only produce the electric signal, and also breathe, under a very specific condition. It is also limited to a single output,, as although these bacteria can usually respire a few different kinds of metals, they use the same pathway to do this, meaning that there would be no way for the bacterium to discern between the two different signals it could produce.</p><br />
<p>The second system relies on phosphatases to remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of cell viability, it also is limited to a single output, as the non-specific phosphatases would act on multiple substrates in a solution. Another issue that could reduce the effectiveness of this system is that there would be background expression of phosphatases in the bacterium as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we turned to a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of metabolism throttling or having to use only a single channel output.</p><br />
<br />
<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
<br />
</html><br />
[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
<html><br />
<br />
<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
<br />
<br><br />
<br />
<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
<br />
<br><br />
<br />
<h2>Genes, Chemicals, and Circuits</h2><br />
<br />
<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
<br />
</html><br />
[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
<html><br />
<br />
<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
<br />
<h2>Does it Work?</h2><br />
<br />
<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
<br />
</html><br />
[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
<html><br />
<br />
<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
<br />
<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
<br />
<h2>What Next?</h2><br />
<br />
<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
<br />
</html>}}}<br />
<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-26T03:57:03Z<p>MaggieRY: </p>
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<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. These compounds have created enormous environmental disturbances, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollution consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). <br />
<br />
Similarly, land and water contaminants often consist of complex mixtures including highly toxic phenols and aromatic compounds, toxic and corrosive carboxylic acids (naphthenic acids) as well as sulfur and nitrogen-containing compounds. These often are recalcitrant, having complex structures that are difficult to break down, causing them to persist in the ecosystem. Classical examples of water contamination include tailings ponds, which contain byproducts from the bitumen extraction process of oil sands. Although the water in tailings ponds is recycled to the extraction process, it is not treated to remove the toxins but are merely contained as much as possible. This creates a susceptibility towards contamination of surrounding areas as a result of these toxic compounds leaching into ground water sources, through spills or through the accidental release of waste products into the environment. </p><br />
<br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>The removal of these compounds is becoming a more and more pressing issue, especially as government bodies start to become more proactive, implementing stricter regulation. Presently, there are a variety of solutions to remove these compounds from the environment using chemical means. These methods involve the use of chemical agents or the physical removal of contaminated soil or water samples and storing these products in contained areas (Scott <i>et al</i>. 2005). There is still however, no efficient, environmentally friendly mechanism for this to occur. The real question is,</p><br />
<br />
<p><b>What do we need in order to remediate these toxins from the environment?</b></p><br />
<br />
<p>We require a method to be able to easily and economically detect where these toxins are and then look to remediating them. Interestingly, microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transforming them into food or other products. Harnessing these natural mechanisms through an engineered synthetic biology could thus be a viable option.</p><br />
<br />
<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
<br />
<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. We illustrated how this sensor could work by showing that it has the potential to detect multiple toxins in contaminated water. Additionally, we developed a miniaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
</p><br />
<p><br />
OSCAR, the Optimized System for Carboxylic Acid Remediation, is designed specifically to target toxins such as naphthenic acids (carboxylic acid-containing compounds), catechol, and nitrogen and sulfur from heterocycles. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol, but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the PetroBrick. When we co-culture these genetic circuits we can selectively produce new compounds from catechol compared to with <i>xylE</i> alone, suggesting that the Petrobrick may be used to create new hydrocarbon based compounds! Lastly we wanted to remove sulfur and nitrogen from heterocycles using the <i>dsz</i> and <i>carA</i> operons respectively. Not only would this improve the quality of fuel produced, but also prevent the production of NO<sub>x</sub> and SO<sub>x</sub> during combustion, reducing the amount of air pollution produced from burning fuel. </p><br />
<br />
<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 200px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">answer a few questions</a> about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
<br />
<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 200px;"></img> in the oilsands? Do they have any concerns about its implementation?</b> We consulted with two professionals working in biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
<br />
<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 200px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
<br />
<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
<br />
<h2>Learn More About FRED and OSCAR</h2><br />
<p>To learn more about our team see the <a href="https://2012.igem.org/Team:Calgary/Project/DataPage">data page</a>, or the <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a> and <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a> overview pages below.</p><br />
<br />
<a href="https://2012.igem.org/Team:Calgary/Project/FRED"><div class="imgbox" id="fredbox"><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/UCalgary2012_EpicBoxFRED_-_Blank.png"></img><br />
</div></a><br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR"><div class="imgbox" id="oscarbox"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/UCalgary2012_EpicBoxOSCAR_-_Blank.png"></img><br />
</div></a><br />
</body><br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-25T22:34:12Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
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<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. These compounds have created enormous environmental disturbances, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollution consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). <br />
<br />
Similarly, land and water contaminants often consist of complex mixtures including highly toxic phenols and aromatic compounds, toxic and corrosive carboxylic acids (naphthenic acids) as well as sulfur and nitrogen-containing compounds. These often are recalcitrant, having complex structures that are difficult to break down, causing them to persist in the ecosystem. Classical examples of water contamination include tailings ponds, which contain byproducts from the bitumen extraction process of oil sands. Although the water in tailings ponds is recycled to the extraction process, it is not treated to remove the toxins but are merely contained as much as possible. This creates a susceptibility towards contamination of surrounding areas as a result of these toxic compounds leaching into ground water sources, through spills or through the accidental release of waste products into the environment. </p><br />
<br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>The removal of these compounds is becoming a more and more pressing issue, especially as government bodies start to become more proactive, implementing stricter regulation. Presently, there are a variety of solutions to remove these compounds from the environment using chemical means. These methods involve the use of chemical agents or the physical removal of contaminated soil or water samples and storing these products in contained areas (Scott <i>et al</i>. 2005). There is still however, no efficient, environmentally friendly mechanism for this to occur. The real question is,</p><br />
<br />
<p><b>What do we need in order to remediate these toxins from the environment?</b></p><br />
<br />
<p>We require a method to be able to easily and economically detect where these toxins are and then look to remediating them. Interestingly, microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transforming them into food or other products. Harnessing these natural mechanisms through an engineered synthetic biology could thus be a viable option.</p><br />
<br />
<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
<br />
<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. We illustrated how this sensor could work by showing that it has the potential to detect multiple toxins in contaminated water. Additionally, we developed a miniaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
</p><br />
<p><br />
OSCAR, the Optimized System for Carboxylic Acid Remediation, is designed specifically to target toxins such as naphthenic acids (carboxylic acid-containing compounds), catechol, and nitrogen and sulfur from heterocycles. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol, but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the PetroBrick. When we co-culture these genetic circuits we can selectively produce new compounds from catechol compared to with <i>xylE</i> alone, suggesting that the Petrobrick may be used to create new hydrocarbon based compounds! Lastly we wanted to remove sulfur and nitrogen from heterocycles using the <i>dsz</i> and <i>carA</i> operons respectively. Not only would this improve the quality of fuel produced, but also prevent the production of NO<sub>x</sub> and SO<sub>x</sub> during combustion, reducing the amount of air pollution produced from burning fuel. </p><br />
<br />
<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 200px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">answer a few questions</a> about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
<br />
<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 200px;"></img> in the oilsands? Do they have any concerns about its implementation?</b> We went to talk to two professionals related to biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
<br />
<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 200px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
<br />
<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
<br />
<h2>Learn More About FRED and OSCAR</h2><br />
<p>To learn more about our team see the <a href="https://2012.igem.org/Team:Calgary/Project/DataPage">data page</a>, or the <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a> and <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a> overview pages below.</p><br />
<br />
<a href="https://2012.igem.org/Team:Calgary/Project/FRED"><div class="imgbox" id="fredbox"><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/UCalgary2012_EpicBoxFRED_-_Blank.png"></img><br />
</div></a><br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR"><div class="imgbox" id="oscarbox"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/UCalgary2012_EpicBoxOSCAR_-_Blank.png"></img><br />
</div></a><br />
</body><br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-25T22:25:47Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
<html><br />
<head><br />
<style><br />
#fredbox{<br />
width: 320px;<br />
height: 215px;<br />
background: #58CD45;<br />
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#oscarbox:hover{<br />
background: #7DD7FF;<br />
}<br />
</style><br />
</head><br />
<body><br />
<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. These compounds have created enormous environmental disturbances, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollution consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). <br />
<br />
Similarly, land and water contaminants often consist of complex mixtures including highly toxic phenols and aromatic compounds, toxic and corrosive carboxylic acids (naphthenic acids) as well as sulfur and nitrogen-containing compounds. These often are recalcitrant, having complex structures that are difficult to break down, causing them to persist in the ecosystem. Classical examples of water contamination include tailings ponds, which contain byproducts from the bitumen extraction process of oil sands. Although the water in tailings ponds is recycled to the extraction process, it is not treated to remove the toxins but are merely contained as much as possible. This creates a susceptibility towards contamination of surrounding areas as a result of these toxic compounds leaching into ground water sources, through spills or through the accidental release of waste products into the environment. </p><br />
<br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>The removal of these compounds is becoming a more and more pressing issue, especially as government bodies start to become more proactive, implementing stricter regulation. Presently, there are a variety of solutions to remove these compounds from the environment using chemical means. These methods involve the use of chemical agents or the physical removal of contaminated soil or water samples and storing these products in contained areas (Scott <i>et al</i>. 2005). There is still however, no efficient, environmentally friendly mechanism for this to occur. The real question is,</p><br />
<br />
<p><b>What do we need in order to better remediate these toxins from the environment?</b></p><br />
<br />
<p>We require a method to be able to easily and economically detect where these toxins are and then look to remediating them. Interestingly, microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transforming them into food or other products. Harnessing these natural mechanisms through an engineered synthetic biology could thus be a viable option.</p><br />
<br />
<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
<br />
<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. We illustrated how this sensor could work by showing that it has the potential to detect multiple toxins in contaminated water. Additionally, we developed a miniaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
</p><br />
<p><br />
OSCAR or our Optimized System for Carboxylic Acid Remediation is designed specifically to target toxins such as naphthenic acids, carboxylic acid-containing compounds, and catechol. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol, but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the PetroBrick. When we co-culture these genetic circuits we can selectively produce new compounds from catechol compared to with <i>xylE</i> alone, suggesting that the Petrobrick may be used to create new hydrocarbon based compounds!<br />
<br />
<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 200px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">answer a few questions</a> about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
<br />
<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 200px;"></img> in the oilsands? Do they have any concerns about its implementation?</b> We went to talk to two professionals related to biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
<br />
<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 200px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
<br />
<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
<br />
<h2>Learn More About FRED and OSCAR</h2><br />
<p>To learn more about our team see the <a href="https://2012.igem.org/Team:Calgary/Project/DataPage">data page</a>, or the <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a> and <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a> overview pages below.</p><br />
<br />
<a href="https://2012.igem.org/Team:Calgary/Project/FRED"><div class="imgbox" id="fredbox"><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/UCalgary2012_EpicBoxFRED_-_Blank.png"></img><br />
</div></a><br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR"><div class="imgbox" id="oscarbox"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/UCalgary2012_EpicBoxOSCAR_-_Blank.png"></img><br />
</div></a><br />
</body><br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-25T17:50:44Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Decatecholization|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<br />
<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006, Schweigert <i>et al.</i>, 2001)). The chemical properties of catechol allow it to react with biomolecules, causing cellular damage including DNA damage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). </p><br />
<p><br />
Catechol is characterized as having a benzene ring with two hydroxyl groups at the 2,3 position. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983).</p><br />
<br />
<p><br />
Currently the registry has two BioBricks available of <i>xylE</i>. One contained <i>xylE</i> with its native ribosome-binding site (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), while the other part contained <i>xylE</i> under the glucose-repressible promoter <i>cstA </i>(<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>xylE</i> expressed by using the <i>tetR</i> promoter (<a href= http://partsregistry.org/Part:BBa_R0040>BBa_R0040</a>).</p> <br />
<br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Figure 1: BioBrick genetic circuit for catechol degradation showing <i>xylE</i> under the ''tetR'' promoter|center]]<html><br />
<h3></h3><br />
<br />
<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of XylE.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Figure 2: Catechol 2,3-dioxygenase (XylE) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu <i>et al</i>., 1995.|center]]<html><br />
<br />
<p>The visual assays were performed with <i>E. coli</i> cells transformed with (<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>) as well as with <i>E. coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >BBa_K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part (<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>) was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was necessary to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011>BBa_K118011</a>). Catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This was indicative that catechol was breaking down into 2-Hydroxymuconate semialdehyde, which was exactly what we expected! This assay was completed by following the protocol written by the 2008 Edinburgh iGEM team.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Figure 3: Results of the catechol visual assay using ''xylE'' [http://partsregistry.org/Part:BBa_K118021 BBa_K118021]. Cultures were grown overnight in LB and the pellets were washed with M9-MM at various times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). Cells were then spun down and catechol was added to the supernatant to 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>xylE</i> gene. The far right tube has <i>E. coli</i> cells without the <i>xylE</i> gene as a negative control and the supernatant remained clear when the catechol was added. |center]]<html><br />
<br />
<a name="Catechol"></a><h2> Converting Catechol into hydrocarbons? </h2><br />
<p>After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our <i>xylE</i> construct (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), we wondered if we could take this any further. What if we could convert this by-product into hydrocarbons too? As catechol is the breakdown product of a number of different degradation pathways in bacteria, this could be particularly useful.</p><br />
<br />
<p>As 2-hydroxymuconate semialdehyde can be further metabolized to pyruvate and acetaldehyde (Harayama S et al., 1987), it seemed possible that these products could be routed into the fatty acid biosynthesis pathway and converted to alkanes using the PetroBrick or the OleT enzyme. Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct could be co-cultured with Petrobrick-containing cells to cooperatively metabolise catechol into hydrocarbons. </p><br />
<br />
<p> In order to test this, we followed this <a href=https://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization>protocol</a>, where we co-cultured cells expressing our <i>xylE</i> construct with either <i>E. coli</i> cells expressing the PetroBrick, or <i>Jeotgalicoccus</i> sp. ATCC 8456 cells expressing OleT. in the presence of catechol.<br />
<br />
<br />
</html>[[File:Calgary PetrobrickCatechol.jpg|600px|thumb|centre|Figure 4: Gas chromatograph of catechol degradation assay using the PetroBrick. While there is limited differences between <i>xylE</i> incubated with and without the PetroBrick, there was one peak with a retention time of 10.5 min which was dramatically increased in the co-culture.]]<html><br />
<br />
</html>[[File:Calgary MSCatecholPetroPeak.jpg|450px|thumb|centre|Figure 5: Mass spectra of the Petrobrick/<i>xylE</i> co-culture retention peak at 10.5 min as shown in Figure 4. While the identity of this compound is currently unknown, there are changes occuring to some of the catechol breakdown products.]]<html><br />
<br />
</html>[[File:Calgary CatechololeTGC.jpg|600px|thumb|centre|Figure 6: Gas chromatograph of catechol degradation assay using <i>Jeotgalicoccus</i> sp. ATCC 8456 a species of bacteria that converts fatty acids into alkenes. This identified a similar peak change in the PetroBrick with a retention time of 10.5 min as shown in Figure 4. This provides additional support that the PetroBrick and this organism can further degrade catechol into breakdown products.]]<html><br />
<br />
</html>[[File:Calgary CatecholMSoleT.jpg|600px|thumb|centre|Figure 7: Mass spectra of <i>Jeotgalicoccus</i> sp. ATCC 8456/<i>xylE</i> co-culture retention peak at 10.5 min as shown in Figure 6. This peak is similar to the peak from the Petrobrick/<i>xylE</i> co-culture, suggesting the breakdown product for both of these cultures is modified catechol from <i>xylE</i>. The identification of this compound is ongoing.]]<html><br />
<br />
<p> Based on our GC-MS results, we were able to show the appearance of a new peak when cells expressing <i>xylE</i> and the PetroBrick were co-cultured. Although we don't know the exact identity of this peak, it is distinct form our control. Interestingly, a similar peak appeared when cells expressing our <i>xylE</i> construct were co-cultured with <i>Jeotgalicoccus</i> sp. ATCC 8456 cells. This suggests that although we don't know the exact identity of this new peak, it is likely that it may be in fact a further breakdown product of catechol. This is a very promising result, as it suggests that in addition to converting naphthenic acids into hydrocarbons, we may also be able to break down catechol, one of the other major toxic components in tailings ponds.</p><br />
<br />
<br />
<br />
<br />
<br />
</html>}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-25T17:25:53Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/TemplateProjectOrange|<br />
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<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. These compounds have created enormous environmental disturbances, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollution consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). <br />
<br />
Similarly, land and water contaminants often consist of complex mixtures including highly toxic phenols and aromatic compounds, toxic and corrosive carboxylic acids (naphthenic acids) as well as sulfur and nitrogen-containing compounds. These often are recalcitrant, having complex structures that are difficult to break down, causing them to persist in the ecosystem. Classical examples of water contamination include tailings ponds, which contain byproducts from the bitumen extraction process of oil sands. Although the water in tailings ponds is recycled to the extraction process, it is not treated to remove the toxins but are merely contained as much as possible. This creates a susceptibility towards contamination of surrounding areas as a result of these toxic compounds leaching into ground water sources, through spills or through the accidental release of waste products into the environment. </p><br />
<br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>The removal of these compounds is becoming a more and more pressing issue, especially as government bodies start to become more proactive, implementing stricter regulation. Presently, there are a variety of solutions to remove these compounds from the environment using chemical means. These methods involve the use of chemical agents or the physical removal of contaminated soil or water samples and storing these products in contained areas (Scott <i>et al</i>. 2005). There is still however, no efficient, environmentally friendly mechanism for this to occur. The real question is,</p><br />
<br />
<p><b>What do we need in order to better remediate these toxins from the environment?</b></p><br />
<br />
<p>We require a method to be able to easily and economically detect where these toxins are and then look to remediating them. Interestingly, microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transforming them into food or other products. Harnessing these natural mechanisms through an engineered synthetic biology could thus be a viable option.</p><br />
<br />
<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
<br />
<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED or our Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. We illustrated how this sensor could work by showing that it has the potential to detect multiple toxins in contaminated water. Additionally, we developed a miniaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
</p><br />
<p><br />
OSCAR or our Optimized System for Carboxylic Acid Remediation is designed specifically to target toxins such as naphthenic acids, carboxylic acid-containing compounds, and catechol. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol, but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the PetroBrick. When we co-culture these genetic circuits we can selectively produce new compounds from catechol compared to with <i>xylE</i> alone, suggesting that the Petrobrick may be used to create new hydrocarbon based compounds!<br />
<br />
<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 200px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">answer a few questions</a> about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
<br />
<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 200px;"></img> in the oilsands? Do they have any concerns about its implementation?</b> We went to talk to two professionals related to biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
<br />
<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 200px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
<br />
<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
<br />
<h2>Learn More About FRED and OSCAR</h2><br />
<p>To learn more about our team see the <a href="https://2012.igem.org/Team:Calgary/Project/DataPage">data page</a>, or the <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a> and <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a> overview pages below.</p><br />
<br />
<a href="https://2012.igem.org/Team:Calgary/Project/FRED"><div class="imgbox" id="fredbox"><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/UCalgary2012_EpicBoxFRED_-_Blank.png"></img><br />
</div></a><br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR"><div class="imgbox" id="oscarbox"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/UCalgary2012_EpicBoxOSCAR_-_Blank.png"></img><br />
</div></a><br />
</body><br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-24T22:55:02Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
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background: #7DD7FF;<br />
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</style><br />
</head><br />
<body><br />
<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. The compounds have created enormous environmental disturbances, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollution consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). <br />
<br />
Similarly, land and water contaminants often consist of complex mixtures including highly toxic phenols and aromatic compounds, toxic and corrosive carboxylic acids (naphthenic acids) as well as sulfur and nitrogen-containing compounds. These often are recalcitrant, having complex structures that are difficult to break down, causing them to persist in the ecosystem. Classical examples of water contamination include tailings ponds, which contain byproducts from the bitumen extraction process of oil sands. Although the water in tailings ponds is recycled to the extraction process, it is not treated to remove the toxins but are merely contained as much as possible. This creates a susceptibility towards contamination of surrounding areas as a result of these toxic compounds leaching into ground water sources, through spills or through the accidental release of waste products into the environment. </p><br />
<br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>The removal of these compounds is becoming a more and more pressing issue, especially as government bodies start to become more proactive, implementing stricter regulation. Presently, there are a variety of solutions to remove these compounds from the environment using chemical means. These methods involve the use of chemical agents or the physical removal of contaminated soil or water samples and storing these products in contained areas (Scott <i>et al</i>. 2005). There is still however, no efficient, environmentally friendly mechanism for this to occur. The real question is,</p><br />
<br />
<p><b>What do we need in order to better remediate these toxins from the environment?</b></p><br />
<br />
<p>We require a method to be able to easily and economically detect where these toxins are and then look to remediating them. Interestingly, microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transforming them into food or other products. Harnessing these natural mechanisms through an engineered synthetic biology could thus be a viable option.</p><br />
<br />
<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
<br />
<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED or our Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. We illustrated how this sensor could work by showing that it has the potential to detect multiple toxins in contaminated water. Additionally, we developed a miniaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
</p><br />
<p><br />
OSCAR or our Optimized System for Carboxylic Acid Remediation is designed specifically to target toxins such as naphthenic acids, carboxylic acid-containing compounds, and catechol. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol, but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the PetroBrick. When we co-culture these genetic circuits we can selectively produce new compounds from catechol compared to with <i>xylE</i> alone, suggesting that the Petrobrick may be used to create new hydrocarbon based compounds!<br />
<br />
<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 200px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">answer a few questions</a> about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
<br />
<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 200px;"></img> in the oilsands? Do they have any concerns about its implementation?</b> We went to talk to two professionals related to biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
<br />
<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 200px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
<br />
<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
<br />
<h2>Learn More About FRED and OSCAR</h2><br />
<p>To learn more about our team see the <a href="https://2012.igem.org/Team:Calgary/Project/DataPage">data page</a>, or the <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a> and <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a> overview pages below.</p><br />
<br />
<a href="https://2012.igem.org/Team:Calgary/Project/FRED"><div class="imgbox" id="fredbox"><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/UCalgary2012_EpicBoxFRED_-_Blank.png"></img><br />
</div></a><br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR"><div class="imgbox" id="oscarbox"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/UCalgary2012_EpicBoxOSCAR_-_Blank.png"></img><br />
</div></a><br />
</body><br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-24T19:09:29Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
<html><br />
<head><br />
<style><br />
#fredbox{<br />
width: 320px;<br />
height: 215px;<br />
background: #58CD45;<br />
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background: #7DD7FF;<br />
}<br />
</style><br />
</head><br />
<body><br />
<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, many toxic compounds are produced. These have created enormous environmental disturbances, burdening our ecosystems with land, water, and air contamination. These toxins consist of a variety of different types of compounds. Air contaminants consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to a variety of environmental issues including green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). Similarly, water contaminants often consist of complex mixtures of compounds including highly toxic phenols and aromatic compounds, corrosive and additionally toxic carboxylic acid containing compounds as well as sulfur and nitrogen-containing compounds. These often have complex structures and cause acute toxicity to wild life. Classical examples of water contamination include tailings ponds produced from the oil extraction process. These are large bodies of water where contaminated supplies are left to settle for long periods of time. Finally, land areas can become contaminated as a result of these toxic compounds leaching into ground water sources, through spills or through the accidental release of waste products into the environment. </p><br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>The removal of these compounds is becoming a more and more pressing issue, especially as government bodies start to become more proactive, implementing stricter regulation. Presently, there are a variety of solutions to remove these compounds from the environment using chemical means. These methods involve the use of chemical agents or the physical removal of contaminated soil or water samples and storing these products in contained areas (Scott <i>et al</i>. 2005). There is still however, no efficient, environmentally friendly mechanism for this to occur. The real question is,</p><br />
<br />
<p><b>What do we need in order to better remediate these toxins from the environment?</b></p><br />
<br />
<p>We require a method to be able to easily and economically detect where these toxins are and then look to remediating them. Interestingly, microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transforming them into food or other products. Harnessing these natural mechanisms through an engineered synthetic biology could thus be a viable option.</p><br />
<br />
<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
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<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED or our Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. We illustrated how this sensor could work by showing that it has the potential to detect multiple toxins in contaminated water. Additionally, we developed a miniaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
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<p><br />
OSCAR or our Optimized System for Carboxylic Acid Remediation is designed specifically to target toxins such as naphthenic acids, carboxylic acid-containing compounds, and catechol. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol, but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the PetroBrick. When we co-culture these genetic circuits we can selectively produce new compounds from catechol compared to with <i>xylE</i> alone, suggesting that the Petrobrick may be used to create new hydrocarbon based compounds!<br />
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<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 200px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">answer a few questions</a> about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
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<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 200px;"></img> in the oilsands? Do they have any concerns about its implementation?</b> We went to talk to two professionals related to biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
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<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 200px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
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<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
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<h2>Learn More About FRED and OSCAR</h2><br />
<p>To learn more about our team see the <a href="https://2012.igem.org/Team:Calgary/Project/DataPage">data page</a>, or the <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a> and <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a> overview pages below.</p><br />
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<a href="https://2012.igem.org/Team:Calgary/Project/FRED"><div class="imgbox" id="fredbox"><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/KillswitchTeam:Calgary/Project/HumanPractices/Killswitch2012-10-04T03:24:17Z<p>MaggieRY: </p>
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<p> The OSCAR component of our project aims to remediate toxins in the oil sands tailings ponds using synthetic bacteria. Despite our belief that the metabolic burden of this system on our bacteria would not allow them to outcompete any native organisms, as we detail in our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">interviews</a> page, our dialogue with experts really emphasized the need to design such a system so as to minimize any escape of our bacteria regardless. As such, we designed a closed <a href=https://2012.igem.org/Team:Calgary/Project/FRED/Prototype>biosensor</a> and a closed <a href=https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor>bioreactor</a> which incorporated built-in structural <a href=https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design> design</a> safety mechanisms. In order to implement one more level of control, which industry felt was needed, we wanted an additional genetic-based containment mechanism to kill our bacteria upon escape from our system, thereby lessening the possibility of OSCAR spreading beyond the bioreactor or horizontally transferring genes to other organisms. We implemented novel ribo-killswitch parts. These contain riboswitch regulatory elements and exo/endonuclease kill genes.</p><br />
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<h2>Click on either element to learn more about it!</h2><br />
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<a style="margin-left: 20px;" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><img src="https://static.igem.org/mediawiki/2012/d/d5/UCalgary2012_FRED_Killswitch_Regulation.png"></img></a> <br />
<a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes"><img src="https://static.igem.org/mediawiki/2012/f/fd/UCalgary2012_OSCAR_Killswitch_KillGene.png"></img></a><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DesulfurizationTeam:Calgary/Project/OSCAR/Desulfurization2012-10-04T03:18:20Z<p>MaggieRY: </p>
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<h2>Why Remove Sulfur?</h2><br />
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<p><br />
Sulfur is the third most abundant element in crude oil (Ma, 2010), and when sulfur containing hydrocarbons are burned they release S0<sub>2</sub> and S0<sub>3</sub> gasses into the atmosphere. Not only does this reduce the efficiency and value of our product, but it also contributes to global warming, acid rain, and various health issues due to the pollution (Reichmuth <i>et al</i>., 2000). Strict regulation on sulfur in fuels are now in place and low-sulfur gasoline is mandated across all of Canada (Source: Environment Canada). To upgrade the quality of our fuel we need to remove the sulfur but keep the hydrocarbon backbone for combustion.</p><br />
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<h2>Our Vision</h2><p><br />
Though a few pathways for biodesulfurization exist in the microbial world, most involve the destruction of part of the carbon skeleton (an example would be the Kodama pathway)(Soleimani <i>et al</i>., 2007). This would effectively reduce the quality of our product. With this in mind the pathway we have chosen is the 4S pathway found in <i>Rhodococcus spp</i>. It has been characterized and shown to remove sulfur from the model substrate dibenzothiophene (DBT) and convert it to 2-hydroxybiphenyl (2-HBP) in a non-destructive manner. DBT and its derivatives make up 70% of the organic sulfur compounds found in crude oil (Ma 2010), and are also some of the most difficult to remove through chemical means. By using the 4S pathway we will be able to upgrade our fuel and remove recalcitrant compounds at the same time. <br />
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</html>[[File:Ucalgary_team_sulfur_4s_enzyme_pathway_diagram.png|center|750px|thumb|Figure 1: The 4S Desulfurization Pathway, showing the desulfurization of the model compound DBT by DszA, DszB, DszC, and DszD.]]<html></p><br />
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<h2>4S pathway</h2><br />
<p><br />
Four enzymes are involved in the 4S pathway, 3 of which are directly involved in the conversion of DBT to 2-HBP. Dibenzothiophene monooxygenase (DszC) is responsible for the first two steps of the pathway, converting DBT to DBT-sulfoxide and finally to DBT-sulfone (DBTO<sub>2</sub>) through the addition of 2 oxygen atoms to the sulfur atom. DBT-sulfone monooxygenase (DszA) then carries out the next step in the pathway, producing 2-hydroxybiphenyl-2-sulfinic acid (HBPS) through addition of a final oxygen to the heteroatom. This causes cleavage of the chemical bonds at the sulfur, breaking the ring and converting the compound from a 3-ring structure to a 2-ring structure. HBPS is then converted to the final product of the 4S pathway by HBPS desulfinase (DszB), producing 2-HBP. At this point, the sulfur has been released from the hydrocarbon in the form of sulfite.</p><p> <br />
The first three steps of the 4S pathway require FMNH<sub>2</sub> and subsequently reduces the reductive power of the cell. WIn order to regain this power an oxidoreductase (DszD) uses NADH to recycle the FMNH<sub>2</sub>, allowing the reaction to proceed. Without DszD the desulfurization pathway would grind to a halt.</p><p align="justify"><br />
The <i>dszA</i>,<i>B</i>, and <i>C</i> genes form an operon on the pSOX plasmid of <i>R. erythropolis</i>, while <i>dszD</i> is found in the chromosome. Naturally this pathway is slow, however using synthetic biology approaches this process can be optimized.</p><br />
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<h2>Our Approach</h2><br />
<h3>1) Find the genes!</h3><br />
<p>We isolated the plasmid containing the <i>dsz</i> genes from a desulfurising environmental isolate of <i>Rhodococcus</i> using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">modified miniprep procedure</a>. As the native promoter has been shown to be repressed by various sulfur-containing compounds (Li <i>et al</i>., 1996), we designed primers for just the coding sequences of the <i>A, B, </i> and <i>C</i> genes. As these genes all have some illegal cutsites in them we constructed them into the PSB1C3 vector and started our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis protocol</a>.</p><br />
<p> We performed an experiment to measure the desulfurization rate of eight compounds by our <i>Rhodococcus</i> strain (figures below). These experiments monitored the degradation of the compounds by our strain over time. We discovered that the <i>dsz</i> operon is capable of desulfurizing a wider range of compounds than just the commonly studied DBT. This shows that this pathway could be a promising solution for degradation of a wide variety of sulfur containing toxins, including those that resemble naphthenic acids. </p> <br />
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<p></html>[[File:Ucalgary2012 DBTGCMS time points.PNG|center|850px|thumb|Figure 2: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS after 6 days of being in the incubator. ]]<html></p<br />
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<p></html>[[File:Ucalgary2012 DBT GCMS.PNG|center|850px|thumb|Figure 3: The peak in this mass spectra demonstrates presence of DBT based on its molecular weight of 184 g/mol. This peak is based on the average of our samples at retention time of 13.9 minute (refer to previous graph).]]<html></p><br />
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<p></html>[[File:Ucalgary2012 DBT GCMS bargraph.PNG|center|650px|thumb|Figure 4: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM DBT with no sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS after 6 days of being in the incubator. Comparing the control with the sample incubated for 6 days shows that the presence of bacteria increases the degradation rate by about ten times.]]<html></p><br />
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<p></html>[[File:Ucalgary2012DesulfurizationTetrahydrdegradation.png|center|600px|thumb|Figure 5: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM compound with no other sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS at the last time point. Degradation is seen, indicating that the pathway has wider substrate specificity than previously thought.]]<html></p><br />
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<p></html>[[File:UcalgaryBenzothiopenecarbozyaldehydedegradation.png|center|600px|thumb|Figure 6: <i>Rhodococcus</i> cells were grown in a modified M9 media containing 0.125mM compound with no other sulfur containing compounds (refer to desulfurization assay protocol for details). Samples were taken out at different time points and were run through GCMS to detect the amount of DBT. The control only contained modified M9 but no bacteria and it was run through the GCMS at the last time point. Degradation is seen, indicating that the pathway has wider substrate specificity than previously thought.]]<html></p><br />
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<h3>2) Mutagenesis: Biobrick Compatability and Increasing DszB Activity </h3><br />
<p>In total the <i>dszABC</i> genes had 7 PstI sites and 1 NotI site that needed to be mutated for the biobrick standard. The primers were designed such that the site was removed without the amino acid being changed. It was also shown that a point mutation changing <i>dszB</i>'s 63rd amino acid from Y to F increases the activity of the protein (Oshiro <i>et al</i>., 2007). This mutation was also included in the mass mutagenesis we undertook. Mutagenesis was performed as per <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">this protocol.</a></p><br />
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<h3>3) Replacing DszD with HpaC & Introducing Catalase </h3><br />
<p><br />
As FMNH<sub>2</sub> is consumed in the first three steps of the pathway it needs to be regenerated or the process will grind to a halt. This usually falls to the <i>dszD</i> gene, however it has been shown that the <i>hpaC</i> gene from <i>E. coli</i> performs the same function more efficiently (Gala´n <i>et al</i>., 2000). One problem arises from this though, as high levels of FMNH<sub>2</sub> cause the production of toxic hydrogen peroxide inside the cell (Gala´n <i>et al</i>. 2000). To address this issue we have included a catalase gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902060"> <i>pLacI-katG-LAA</i></a>) that will remove the peroxide that would be toxic to the cell.</p><br />
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<p></html>[[File:Ucalgary_sulfur_constructs_KatandHpaC.PNG|center|250px|thumb|Figure 7: Diagrammatic representation of the full "optimization circuit", consisting of the oxidoreductase HpaC and a catalase (KatG).]]<html></p><br />
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<h3>Results</h3><br />
<p>To show that catalase activity increased <i>E. coli</i> survivability in peroxide we cultured the inducible catalase against a catalase-free control with varying levels of peroxide. After growing overnight the negative didn't grow in any culture except in the absence of peroxide, while the catalase cultures could tolerate peroxide. This is shown below.</p><p><br />
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</html>[[File:J04500-K137068 KatG assay sulfurucalgary.png|center|600px|thumb|Figure 8: Catalase Assay. Overnight cultures of pLacI and pLacI-KatGLAA were innoculated into 0 mM, 1 mM, 5 mM, and 10 mM peroxide. Cultures were grown overnight and turbidity was observed. It was found that at 1 mM of peroxide, cultures with just the lacI promotor perished, however when KatG-LAA was expressed, the cells survived.]]<html></p><br />
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<p>To test the action of HpaC to use NADH to recycle FMN into FMNH<sub>2</sub> cell lysates were exposed to NADH and it's absorbance at 340nm (Kamali <i>et al</i>., 2010) was measured over time. Both native HpaC expression and an induced <a href="http://partsregistry.org/Part:BBa_K902058"><i>pLacI-RBS-hpaC</i></a> system were tested as well as a negative control. The results are shown below.</p><br />
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<p> </html> <br />
[[File:Ucalgary2012 HpaC assaycumulativeforthedatapage.png|center|850px|thumb|Figure 9: HpaC Assay with '''A)''' 2 mL cell lysate and '''B)''' 100 &micro;L cell lysate. Cultures of pLacI-hpaC and pLacI-dszB were grown up overnight in LB with appropriate antibiotics. The following morning, cells were subcultured 1/4 into LB with 200 &micro;M IPTG and allowed to grow for 2h in order to induce protein expression. 1 mL samples of cells were then transferred to 2 mL tubes, washed twice in 50 mM Tris-HCl (pH 7.5) and resuspended in this buffer. Samples were then subjected to 5 freeze-thaw cycles in order to lyse cells. After spinning down samples, various amounts of cell lysate were transferred to a cuvette, and a spectrophotometer was blanked at 340 nm with this sample. 140 &micro;M NADH and 20 &micro;M FMN was then added, the cuvette was quickly inverted, and readings were taken at 340 nm. pLacI-dszB was used as a control to measure native amounts of oxidoreductase activity, whereas the pLacI-hpaC cultures were used to measure activity when HpaC was expressed. The control was just Tris-HCl buffer with the NADH and FMN compounds added. Decrease in absorbance at 340 nm corresponds to the loss of NADH as it is converted to NAD+.]]<html></p><br />
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<p>The assay showed that NADH does not abiotically convert into NAD+, however the native expression of HpaC did show a steady decrease in the levels of NADH. The induced overexpression of HpaC caused extremely rapid conversion into NAD+ as reflected by a sharp drop in the absorbance of NADH (see figure B). This drop was much sharper than what was seen when native levels of oxidoreductases were tested, showing that the <a href="http://partsregistry.org/Part:BBa_K902058"><i>pLacI-RBS-hpaC</i></a> was functional and that it would effectively recycle FMN.</p><br />
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<h3>4) Optimizing Gene Order</h3><br />
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<p>Further optimization of the system was achieved through reorganization of the reconstructed operon. Natively the genes are arranged ABC, however the catalytic efficiency of the protein products are 25:1:5 for A:B:C respectively (Li <i>et al</i>., 2008). By rearranging the genes into BCA there is stronger transcription of the weaker proteins, giving a more balanced system overall. These would all be constructed with the same strong ribosomal binding site, <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>.</p><br />
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</html>[[File:DszOperonOptimize.png|center|400px|thumb|Figure 10: Method of optimizing gene order. The top circuit represents that found natively in the organism, with the bottom circuit representing our modified version.]]<html><br />
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<h2>Final Constructs</h2><br />
<p>After all of the above considerations are met, four final constructs for our system will be made to allow us to test desulfurization under different conditions.</p><p><br />
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</html>[[File:WikiConstructs_ucalgary_sulfur_2012_final_systems.png|center|700px|thumb|Figure 11: First set of final constructs for the desulfurization operon, with constitutive Dsz expression and inducible expression of the optimization proteins; either HpaC on its own or coexpressed with KatG]]<html></p><br />
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<p><br />
The first two constructs have the modified <i>dsz</i> operon (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902052"><i>dszB</i></a>, <i>dszC</i>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902050"><i>dszA</i></a>) under the control of a constitutive TetR promotor (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>) This is to allow for the testing of the optimization circuit, which is under the control of a lacI promotor inducible by IPTG (<a href="http://partsregistry.org/Part:BBa_J04500">BBa_J04500</a>). The set-up of these two constructs will therefore allow for the expression of the <i>dsz</i> genes with the ability to test and compare their desulfurization rates <br> A) On their own <br> B) With the addition of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> <br> C) With the addition of both <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902057"><i>hpaC</i></a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K137068"><i>katG-LAA</i></a></p><br />
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<p>This will allow us to determine what the optimal construct and expression levels of the additional genes must be in order to have the most effective sulfur removal system.</p><br />
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</html>[[File:WikiConstructs2 sulfur ucalgary induciblesytems.PNG|center|700px||thumb|Figure 12: Second set of final constructs for the desulfurization operon, with all genes under an IPTG inducible promotor.]]<html><br />
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<p><br />
Due to the large number of proteins being expressed in this system, the possibility of forming inclusion bodies is present. As such, a backup system was built where both the optimization circuit and the <i>dsz</i> operon were under the control of the inducible lacI promoter. This system would allow us to tune the expression of the genes, and determine which expression level is optimal for desulfurization in our bioreactor.</p> <br />
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<p>Currently, assembly of these final constructs is underway, with only a couple more construction steps before functionality tests can begin.</p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-04T02:59:11Z<p>MaggieRY: </p>
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This year, our team wanted to identify a novel responsive element capable of detecting and quantifying tailings ponds toxins (e.g. naphthenic acids/NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. naphthenic acids/NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
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<h2>Transposons: What, How, Why?</h2><br />
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The transposable element (TE), Tn5, is a conservative transposon that is able to insert a segment of genes bordered by specific 19bp insertion sequences (IS) from one part of the genome (e.g. plasmid vector) randomly to another location, such as the chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>Tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
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</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
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<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
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Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
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<h2>Toxin-Degrading Organism Used</h2><br />
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<i>Pseudomonads</i> have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón et al. 2010; Herman et al., 1994; Del Rio et al., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins. We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens pf-5</i> strain is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, sequencing data is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
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<br />
<br><br />
<br />
<br />
<h2>Method Design</h2><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 1: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens pf-5</i> using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers et al., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman&Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h2>Detection by Mutant <i>Pseudomonas fluorescens</i> Pf-5</h2><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 2Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 3: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 5: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 6: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br><br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">In order to determine which genes the transposon has inserted into, the self-cloning properties of the transposon were utilized. By digesting the genome, religating, and transforming <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. We are still awaiting the sequencing results for these, however results so far are a promising step towards a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. In tandem as we await sequencing results, our next steps will be to test these strains in conjunction with our electrochemical detector.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-04T02:55:48Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img><br />
<p align="justify"><br />
This year, our team wanted to identify a novel responsive element capable of detecting and quantifying tailings ponds toxins (e.g. naphthenic acids/NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. naphthenic acids/NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.<br />
</p><br />
<h2>Transposons: What, How, Why?</h2><br />
<p align="justify"><br />
The transposable element (TE), Tn5, is a conservative transposon that is able to insert a segment of genes bordered by specific 19bp insertion sequences (IS) from one part of the genome (e.g. plasmid vector) randomly to another location, such as the chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>Tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p><br />
<br />
</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html><br />
<br />
<br />
<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p><br />
<p align="justify"><br />
Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.<br />
</p><br />
<br />
<br />
<br><br />
<h2>Toxin-Degrading Organism Used</h2><br />
<p align="justify"><br />
<i>Pseudomonads</i> have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón et al. 2010; Herman et al., 1994; Del Rio et al., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins. We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens pf-5</i> strain is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, sequencing data is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.<br />
</p><br />
<br />
<br><br />
<br />
<br />
<h2>Method Design</h2><br />
<p align="justify"><br />
To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p><br />
<br />
<p align="justify"><br />
</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 1: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an<br />
<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html><br />
</p><br />
<br />
<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens pf-5</i> using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers et al., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p><br />
<br />
<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p><br />
</p><br />
<br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman&Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzymes that do not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p><br />
<br />
<p align="justify">For a detailed protocol, please consult our <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p><br />
<br />
<h2>Results</h2><br />
<h2>Detection by Mutant <i>Pseudomonas fluorescens</i> Pf-5</h2><br />
<br />
<br />
<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screening to test the specificity of their responses.</p><br />
<br />
<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 2Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16h. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify"><br />
<br />
<br />
Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.<br />
</p><br />
<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 3: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 5: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br />
<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 6: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p><br />
<br><br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">In order to determine which genes the transposon has inserted into, the self-cloning properties of the transposon were utilized. By digesting the genome, religating, and transforming <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. We are still awaiting the sequencing results for these, however results so far are a promising step towards a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. In tandem as we await sequencing results, our next steps will be to test these strains in conjunction with our electrochemical detector.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T01:29:26Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable, was a fed-batch closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings in a continuous stir method, which are favored for an industrial scale of (1000+ L tanks). Product is removed at the same rate that biomass and nutrients are added, and tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p><br />
<br />
<p>Lastly, we needed to pick the best possible process to remove the hydrocarbons from the culture. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. <br />
<br />
To ensure that the belt does not transfer live bacteria into the hydrocarbon tank, we will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of light before the hydrocarbons are skimmed from the bioreactor chamber.<br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. In the events when we were using live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
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<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
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<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
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<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel and ensuring even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for our aerobic bacteria to thrive. When assembled together the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger; a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Lastly, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<p> Furthermore, we tested our belts ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test for us since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, there are many different types of NA's in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This data suggests that minimal NA's were found in our skimmed hydrocarbon layer and that most were left in the water layer. <br />
</html>[[File:HC layer, skimmed (no NaOH)-ucalgary.png|thumb|300px|left|'''Figure 4:''' This graph shows the amount of NA's found in the skimmed hydrocarbon layer. Each bar represents the carbon ring count for a different type of NA. Clearly, minimal NA's were skimmed into the hydrocarbon layer.]]<html><br />
</html>[[File:Water layer, skimmed-ucalgary.png|thumb|300px|centre|'''Figure 5:''' This graph shows how many NA's were left in the water layer of our skimmed solution. This data suggests that minimal amounts of NA were skimmed into our solution, and with most of those skimmed found in the water layer.]] <html><br />
<br />
<br />
<h2>The Final System</h2><br />
<p>Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR bacterium would have the most suitable kill-switch circuit attached to its respective hydrocarbon conversion circuit. We envision OSCAR to be a co-culture of decarboxylation, decatecholization, denitrogenation, and desulfurization. Lastly, due to the energetically expensive nature of maintaining the circuits, we anticipate that if the circuits are constitutively produced cell growth rate may be very slow. Therefore in the final circuits we would want them to be activated by quorum sensing promoter systems. Essentially, when cells are at low density they focus energy on growth; when cells reach appropriate density for the reaction chamber, transcription of the circuit is enabled. Together we hope that the system will clean up recalcitrant petroleum waste and produce energy.<br />
<br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T01:06:31Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable, was a fed-batch closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings in a continuous stir method, which are favored for an industrial scale of (1000+ L tanks). Product is removed at the same rate that biomass and nutrients are added, and tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p><br />
<br />
<p>Lastly, we needed to pick the best possible process to remove the hydrocarbons from the culture. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. <br />
<br />
To ensure that the belt does not transfer live bacteria into the hydrocarbon tank, will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of light before the hydrocarbons are skimmed from the bioreactor chamber.<br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. In the events when we were using live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel and ensuring even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for our aerobic bacteria to thrive. When assembled together the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger; a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Lastly, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<p> Furthermore, we tested our belts ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test for us since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, there are many different types of NA's in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This suggests that minimal NA's were skimmed into our hydrocarbon layer and that most were left in the water layer. <br />
<br />
<br />
<h2>The Final System</h2><br />
Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR would have the most suitable kill-switch circuit attached <br />
<br />
<br />
<h2>Future Directions</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, pH, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial growth and production cycle. Since we would prefer that our bacteria produce hydrocarbons within their stationary phase, we would like to look into controlling the genetic circuits to become active during this portion of the life-cycle (transcriptional activation coupled to quorum sensing) and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also because we envision OSCAR to be a co-culture of decarboxylation, decatecholizatoin, denitrogenation, and desulfurization, we will want to test the effectiveness of this co-culturing system to produce hydrocarbons and the ability of our prototype to selectively extract them.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T00:57:35Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable, was a fed-batch closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings in a continuous stir method, which are favored for an industrial scale of (1000+ L tanks). Product is removed at the same rate that biomass and nutrients are added, and tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p><br />
<br />
<p>Lastly, we needed to pick the best possible process to remove the hydrocarbons from the culture. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. <br />
<br />
To ensure that the belt does not transfer live bacteria into the hydrocarbon tank, will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of light before the hydrocarbons are skimmed from the bioreactor chamber.<br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. In the events when we were using live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel and ensuring even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for our aerobic bacteria to thrive. When assembled together the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger; a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Lastly, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<p> Furthermore, we tested our belts ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test for us since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, there are many different types of NA's in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. <br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T00:46:00Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable, was a fed-batch closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings in a continuous stir method, which are favored for an industrial scale of (1000+ L tanks). Product is removed at the same rate that biomass and nutrients are added, and tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p><br />
<br />
<p>Lastly, we needed to pick the best possible process to remove the hydrocarbons from the culture. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. <br />
<br />
To ensure that the belt does not transfer live bacteria into the hydrocarbon tank, will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of light before the hydrocarbons are skimmed from the bioreactor chamber.<br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. Like our concept for the scaled up design, we made our prototype a closed system to prevent cross contamination with microbes outside the chamber. We also optimized belt skimmers with vegetable oil and water, optimizing which belts materials were most suitable for our application. Differing from the large-scale design however, we needed to used a batch system for the lab scale to optimize appropriate growth conditions, for practical considerations. </p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>To optimize growth conditions we tested rate of growth in cultures that did and did not include a sparger and a turbine. The purpose of the turbine is to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel and ensuring even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T00:41:27Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable, was a fed-batch closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings in a continuous stir method, which are favored for an industrial scale of (1000+ L tanks). Product is removed at the same rate that biomass and nutrients are added, and tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p><br />
<br />
<p>Lastly, we needed to pick the best possible process to remove the hydrocarbons from the culture. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. <br />
<br />
To ensure that the belt does not transfer live bacteria into the hydrocarbon tank, will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of light before the hydrocarbons are skimmed from the bioreactor chamber.<br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. Like our concept for the scaled up design, we made our prototype a closed system to prevent cross contamination with microbes outside the chamber. We also optimized belt skimmers with vegetable oil and water with scaled down size. Differing from the large-scale design however, we needed to used a batch system for the lab scale to optimize appropriate growth conditions, for practical considerations. </p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>To optimize growth conditions we tested rate of growth in cultures that did and did not include a sparger and a turbine. The purpose of the turbine is to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel and ensuring even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T00:31:40Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable, was a fed-batch closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings in a continuous stir method, which are favored for an industrial scale of (1000+ L tanks). Product is removed at the same rate that biomass and nutrients are added, and tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p><br />
<br />
<p>Lastly, we needed to pick the best possible process to remove the hydrocarbons from the culture. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. <br />
<br />
To ensure that the belt does not transfer live bacteria into the hydrocarbon tank, will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of light before the hydrocarbons are skimmed from the bioreactor chamber.<br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a the essential concepts that needed to be developed in the prototype. We need to make our bioreactor a closed system in order to prevent it from becoming contaminated. We needed to used a batch system for the lab scale (for competition data) of optimizing appropriate growth media. This opposes </p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T00:24:23Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable, was a fed-batch system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings in a continuous stir method. Product is removed at the same rate that biomass and nutrients are added. A continuous method would be more appropriate at an industrial scale (1000+ L tanks), where the bacteria will have enough time to convert toxins in such large tanks. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p><br />
<br />
<br />
<p>Lastly, we needed a process to remove the hydrocarbons once they were produced. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. <br />
<br />
To ensure that the belt does not transfer live bacteria into the hydrocarbon tank, will have a UV light attached across the belt to ensure sterilization of any bacteria picked up by the skimmer before the hydrocarbons are skimmed from the bioreactor chamber.<br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a the essential concepts that needed to be developed in the prototype. We need to make our bioreactor a closed system in order to prevent it from becoming contaminated. We needed to used a batch system for the lab scale (for competition data) of optimizing appropriate growth media. This opposes </p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T00:09:57Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to considered in big systems: open or closed (theirs was not contained), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors researching bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Below are pictures from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. Our ideal process would be a fed-batch system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. The fresh tailing would be added when the toxin concentration within the bioreactor is lower, thus allowing the bacteria to continually convert toxins to hydrocarbons. Additionally, the process would have to occur within an enclosed system to ensure its sterility.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a few basic concepts that needed to be included in the prototype. For one, we needed to make our bioreactor a closed system in order to keep our solution sterile. This is necessary to allow only our bacteria (<i>E. coli</i> with an ampicillin resistance marker) to grow. Employing a closed system bioreactor is also necessary as a physical containment measure to confine our organisms to our system.</p><br />
<p>Next, we needed to use a batch system for our system to work at lab scale (for competition data). This means that we are allowing the entire process to occur, and then removing all of our solution when our reactions have come to completion. This batch will include an appropriate growth medium to optimize our organisms growth, as well as a compound. This opposes a continuous stir method, where product is removed at the same rate that biomass and nutrients are added. A continuous method would be more appropriate at an industrial scale (1000+ L tanks), where the bacteria will have enough time to convert toxins in such large tanks. </p><br />
<p>Furthermore, we had to find a way to remove the hydrocarbons once they were produced. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method is useful because it allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. To ensure sterility the belt skimmer will have a UV light attached across the belt to ensure sterilization of any bacteria picked up by the skimmer before the hydrocarbons are skimmed from the bioreactor chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T00:06:24Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we researched papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery), interviewed plant managers and learned conditions that need to considered in big systems: open or closed, mechanisms of oxygenation and preventing settling and containment (unlike our system, theirs is open since they use unmodified microbes to do the work. We also interviewed graduate students researching bioreactors at the University of Calgary for their insight into the application that we would like to try and lastly had weekly meetings with the supervisors and biologists on our team. Below are pictures from our trip!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. Our ideal process would be a fed-batch system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. The fresh tailing would be added when the toxin concentration within the bioreactor is lower, thus allowing the bacteria to continually convert toxins to hydrocarbons. Additionally, the process would have to occur within an enclosed system to ensure its sterility.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a few basic concepts that needed to be included in the prototype. For one, we needed to make our bioreactor a closed system in order to keep our solution sterile. This is necessary to allow only our bacteria (<i>E. coli</i> with an ampicillin resistance marker) to grow. Employing a closed system bioreactor is also necessary as a physical containment measure to confine our organisms to our system.</p><br />
<p>Next, we needed to use a batch system for our system to work at lab scale (for competition data). This means that we are allowing the entire process to occur, and then removing all of our solution when our reactions have come to completion. This batch will include an appropriate growth medium to optimize our organisms growth, as well as a compound. This opposes a continuous stir method, where product is removed at the same rate that biomass and nutrients are added. A continuous method would be more appropriate at an industrial scale (1000+ L tanks), where the bacteria will have enough time to convert toxins in such large tanks. </p><br />
<p>Furthermore, we had to find a way to remove the hydrocarbons once they were produced. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method is useful because it allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. To ensure sterility the belt skimmer will have a UV light attached across the belt to ensure sterilization of any bacteria picked up by the skimmer before the hydrocarbons are skimmed from the bioreactor chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-03T23:57:35Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similar sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we read research papers on bioreactors, toured a wastewater treatment plant, interviewed graduate students in the field, and had weekly meetings with the supervisors and biologists on our team. The bioreactors at the wastewater treatment plant were contained in open systems since they used natural microbes. The reactor also contained an air sparger to oxygenate the solution and spun at an extraordinarily slow rate. This was one of the many bioreactor processes we looked into. Below are pictures from our trip!</p><br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. Our ideal process would be a fed-batch system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. The fresh tailing would be added when the toxin concentration within the bioreactor is lower, thus allowing the bacteria to continually convert toxins to hydrocarbons. Additionally, the process would have to occur within an enclosed system to ensure its sterility.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a few basic concepts that needed to be included in the prototype. For one, we needed to make our bioreactor a closed system in order to keep our solution sterile. This is necessary to allow only our bacteria (<i>E. coli</i> with an ampicillin resistance marker) to grow. Employing a closed system bioreactor is also necessary as a physical containment measure to confine our organisms to our system.</p><br />
<p>Next, we needed to use a batch system for our system to work at lab scale (for competition data). This means that we are allowing the entire process to occur, and then removing all of our solution when our reactions have come to completion. This batch will include an appropriate growth medium to optimize our organisms growth, as well as a compound. This opposes a continuous stir method, where product is removed at the same rate that biomass and nutrients are added. A continuous method would be more appropriate at an industrial scale (1000+ L tanks), where the bacteria will have enough time to convert toxins in such large tanks. </p><br />
<p>Furthermore, we had to find a way to remove the hydrocarbons once they were produced. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method is useful because it allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. To ensure sterility the belt skimmer will have a UV light attached across the belt to ensure sterilization of any bacteria picked up by the skimmer before the hydrocarbons are skimmed from the bioreactor chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-03T23:39:12Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system at the scale to operate in the locations of oil sands tailings ponds and oil refineries. We drew inspiration from literature and what is known of similar sized bioreactors used for other applications (waste-water treatment plants, tissue engineering, and beer fermentation) to decide what factors to consider in the design of OSCAR's home. </p><br />
<p>We were also tasked with finding an optimal method for extracting the hydrocarbon products from the bioreactor. The hydrophobicity and light density of the hydrocarbons should cause them to fraction into the top layer of the solution. Several methods for separation were considered to maintain layer separation into their respective components such as centrifugation or filtration.</p> <br />
<p>In light of the issue of containment with genetically modified organisms, we have designed our system with several levels of controls that range from biological to physical containment measures to keep our organisms contained. The bacteria we are working with in our system are nonpathogenic, lab strains of <i>Escherichia coli</i> transformed with genes that are found in naturally occurring tailings pond organisms. Although considered harmless, it is preferable to prevent the OSCAR bacteria from escaping into the environment. We have implemented containment controls into the physical design and biological controls in the form of a <a href=https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch>genetic kill switch</a><br />
mechanism. </p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we read research papers on bioreactors, toured a wastewater treatment plant, interviewed graduate students in the field, and had weekly meetings with the supervisors and biologists on our team. The bioreactors at the wastewater treatment plant were contained in open systems since they used natural microbes. The reactor also contained an air sparger to oxygenate the solution and spun at an extraordinarily slow rate. This was one of the many bioreactor processes we looked into. Below are pictures from our trip!</p><br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. Our ideal process would be a fed-batch system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. The fresh tailing would be added when the toxin concentration within the bioreactor is lower, thus allowing the bacteria to continually convert toxins to hydrocarbons. Additionally, the process would have to occur within an enclosed system to ensure its sterility.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a few basic concepts that needed to be included in the prototype. For one, we needed to make our bioreactor a closed system in order to keep our solution sterile. This is necessary to allow only our bacteria (<i>E. coli</i> with an ampicillin resistance marker) to grow. Employing a closed system bioreactor is also necessary as a physical containment measure to confine our organisms to our system.</p><br />
<p>Next, we needed to use a batch system for our system to work at lab scale (for competition data). This means that we are allowing the entire process to occur, and then removing all of our solution when our reactions have come to completion. This batch will include an appropriate growth medium to optimize our organisms growth, as well as a compound. This opposes a continuous stir method, where product is removed at the same rate that biomass and nutrients are added. A continuous method would be more appropriate at an industrial scale (1000+ L tanks), where the bacteria will have enough time to convert toxins in such large tanks. </p><br />
<p>Furthermore, we had to find a way to remove the hydrocarbons once they were produced. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method is useful because it allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. To ensure sterility the belt skimmer will have a UV light attached across the belt to ensure sterilization of any bacteria picked up by the skimmer before the hydrocarbons are skimmed from the bioreactor chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-03T23:31:59Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>A major goal of our project is to use genetically engineered bacteria to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team needed to design a contained bioreactor system to house these conversions. Because of the enormous scale of the oil sands tailings ponds and refineries, we needed to design our vessel considering these applications. We drew inspiration from literature and what is known of similar sized bioreactors used for other applications (waste-water treatment plants, tissue engineering, and beer fermentation) to decide what factors to consider in the design of OSCAR's home. </p><br />
<p>We were also tasked with finding an optimal method for extracting the hydrocarbon products from the bioreactor. The hydrophobicity and light density of the hydrocarbons should cause them to fraction into the top layer of the solution. Several methods for separation were considered to maintain layer separation into their respective components such as centrifugation or filtration.</p> <br />
<p>In light of the issue of containment with genetically modified organisms, we have designed our system with several levels of controls that range from biological to physical containment measures to keep our organisms contained. The bacteria we are working with in our system are nonpathogenic, lab strains of <i>Escherichia coli</i> transformed with genes that are found in naturally occurring tailings pond organisms. Although considered harmless, it is preferable to prevent the OSCAR bacteria from escaping into the environment. We have implemented containment controls into the physical design and biological controls in the form of a <a href=https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch>genetic kill switch</a><br />
mechanism. </p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we read research papers on bioreactors, toured a wastewater treatment plant, interviewed graduate students in the field, and had weekly meetings with the supervisors and biologists on our team. The bioreactors at the wastewater treatment plant were contained in open systems since they used natural microbes. The reactor also contained an air sparger to oxygenate the solution and spun at an extraordinarily slow rate. This was one of the many bioreactor processes we looked into. Below are pictures from our trip!</p><br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. Our ideal process would be a fed-batch system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. The fresh tailing would be added when the toxin concentration within the bioreactor is lower, thus allowing the bacteria to continually convert toxins to hydrocarbons. Additionally, the process would have to occur within an enclosed system to ensure its sterility.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a few basic concepts that needed to be included in the prototype. For one, we needed to make our bioreactor a closed system in order to keep our solution sterile. This is necessary to allow only our bacteria (<i>E. coli</i> with an ampicillin resistance marker) to grow. Employing a closed system bioreactor is also necessary as a physical containment measure to confine our organisms to our system.</p><br />
<p>Next, we needed to use a batch system for our system to work at lab scale (for competition data). This means that we are allowing the entire process to occur, and then removing all of our solution when our reactions have come to completion. This batch will include an appropriate growth medium to optimize our organisms growth, as well as a compound. This opposes a continuous stir method, where product is removed at the same rate that biomass and nutrients are added. A continuous method would be more appropriate at an industrial scale (1000+ L tanks), where the bacteria will have enough time to convert toxins in such large tanks. </p><br />
<p>Furthermore, we had to find a way to remove the hydrocarbons once they were produced. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method is useful because it allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. To ensure sterility the belt skimmer will have a UV light attached across the belt to ensure sterilization of any bacteria picked up by the skimmer before the hydrocarbons are skimmed from the bioreactor chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-03T23:25:16Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
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<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>A major goal of our project was to use genetically engineered bacteria to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team needed to create a contained bioreactor system to carry out these conversions. Because of the large scale of the oil sands tailings ponds, we needed to design our vessel for large scale application. Similar sized bioreactors can be found in waste-water treatment plants, tissue engineering, and beer fermentation. All these types of systems need highly controlled heat and oxygen exchange,suitable pH and agitation control. For our system we had the additional design considerations of determining ideal flow rates, growth medium, and optimal conditions for bacterial growth and biochemical conversion. </p><br />
<p>In addition to developing a design for the bioreactor taking these factors into account, we were also tasked with finding a way of extracting the hydrocarbon products from the bioreactor. The hydrophobicity and light density of the hydrocarbons should cause them to fraction into the top layer of the solution. Several methods for separation were considered to maintain layer separation into their respective components such as centrifugation or filtration.</p> <br />
<p>The bacteria we are working with in our system are nonpathogenic, lab strains of <i>Escherichia coli</i> transformed with genes that are found in naturally occurring tailings pond organisms. Although they are considered harmless, it is preferable to prevent their escape into the environment. We have implemented containment controls into the design and biological controls in the form of a <a href=https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch>genetic kill switch</a><br />
mechanism. In light of the issue of containment with genetically modified organisms, we have designed our system with several levels of controls that range from biological to physical containment measures to keep our organisms contained. </p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|'''Figure 1:''' Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the plants bioreactors. ]]<html><br />
<p>Before diving into a making bioreactor, we first had to research current solutions in the field. To help us with this phase, we read research papers on bioreactors, toured a wastewater treatment plant, interviewed graduate students in the field, and had weekly meetings with the supervisors and biologists on our team. The bioreactors at the wastewater treatment plant were contained in open systems since they used natural microbes. The reactor also contained an air sparger to oxygenate the solution and spun at an extraordinarily slow rate. This was one of the many bioreactor processes we looked into. Below are pictures from our trip!</p><br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. Our ideal process would be a fed-batch system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. The fresh tailing would be added when the toxin concentration within the bioreactor is lower, thus allowing the bacteria to continually convert toxins to hydrocarbons. Additionally, the process would have to occur within an enclosed system to ensure its sterility.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|'''Figure 2:''' From computer to prototype, how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took its shape from the materials we were able to find in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is our first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype</h2><br />
<p>We determined a few basic concepts that needed to be included in the prototype. For one, we needed to make our bioreactor a closed system in order to keep our solution sterile. This is necessary to allow only our bacteria (<i>E. coli</i> with an ampicillin resistance marker) to grow. Employing a closed system bioreactor is also necessary as a physical containment measure to confine our organisms to our system.</p><br />
<p>Next, we needed to use a batch system for our system to work at lab scale (for competition data). This means that we are allowing the entire process to occur, and then removing all of our solution when our reactions have come to completion. This batch will include an appropriate growth medium to optimize our organisms growth, as well as a compound. This opposes a continuous stir method, where product is removed at the same rate that biomass and nutrients are added. A continuous method would be more appropriate at an industrial scale (1000+ L tanks), where the bacteria will have enough time to convert toxins in such large tanks. </p><br />
<p>Furthermore, we had to find a way to remove the hydrocarbons once they were produced. We decided to use a belt skimmer, similar to those used to help clean up oil spills. This method is useful because it allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons. To ensure sterility the belt skimmer will have a UV light attached across the belt to ensure sterilization of any bacteria picked up by the skimmer before the hydrocarbons are skimmed from the bioreactor chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Finally to ensure optimal bacterial growth and production of our desired product, we included both a sparger and a turbine in the bioreactor design. The purpose of the turbine was to mix the solution preventing bacterial cells and other heavier materials from settling at the bottom of the vessel. Continuous mixing of the solution would also ensure even nutrient and reactant distribution in the tank. The sparger oxygenated the solution, aeration which is necessary for our aerobic bacteria to thrive. When assembled together the turbine would be located above the sparger thus breaking each bubble from the sparger into smaller ones. In a thicker solution such as tailings, both the air sparger and turbine will agitate the solution thus mixing it and prevent heavier materials from settling at the bottom of the chamber.</p><br />
<br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using programs called Maya and RealFlow. Maya is a complex and extremely versatile computer animation program used for many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. In addition to this, we made a functioning model of a tank with our turbine, belt skimmer, and sparger included inside. Our model is also a closed system, which will be brought to the competition to demonstrate to the judges.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/N7phu6NmlQo" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), so we conducted three different tests to determine which belt would work best for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|'''Figure 3:''' This table represents the belt rankings of three different tests. We tested for the ability of the belts to pick up hydrocarbons, and the abilities of the belts to not pick up bacteria or tailings pond solution. Our five tested belts and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The test was conducted with a turbine and a sparger, only a turbine, and only a sparger. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|'''Figure 4:''' This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was very encouraging data since we want to skim our produced hydrocarbons from the top layer of our bioreactor.]]<html><br />
<br />
<p>Also, we mixed water, commercial NA’s, and hexadecane (model hydrocarbon) together in a small test tube to determine if we will indeed get a top layer of hydrocarbons like what we need. Indeed, this top layer of hydrocarbons was formed after two minutes of time to allow for separation.</p><br />
<br />
<br />
<h2>The Final System</h2><br />
<br />
<h2>Next Steps</h2><br />
<p>The next steps would include developing a feedback and sensing method to monitor the temperature, Ph, and C02 in a highly toxic and corrosive environment. Once the genetically engineered bacterium is produced we can start modeling the bacterial life cycle. Since our bacteria will only produce hydrocarbons within its stationary phase, we would like to look into increasing this portion of the lifecycle and possibly into adding bacteria to the bioreactor when it is in its exponential phase rather than the lag phase. This should reduce the time needed for each reactor cycle. Also, another important aspect we can test after the bacteria are made is the rate of hydrocarbon production by the bacteria.</p><br />
<br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-03T22:38:16Z<p>MaggieRY: </p>
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<br />
<br />
<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006, Schweigert <i>et al.</i>, 2001)). The chemical properties of catechol allow it to react with biomolecules, causing cellular damage including DNA damage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). </p><br />
<p><br />
Catechol is characterized as having a benzene ring with two hydroxyl groups at the 2,3 position. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983).</p><br />
<br />
<p><br />
Currently the registry has two BioBricks available of <i>xylE</i>. One contained <i>xylE</i> with its native ribosome-binding site (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), while the other part contained <i>xylE</i> under the glucose-repressible promoter <i>cstA </i>(<a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>xylE</i> repressed by using the TetR promoter (<a href= http://partsregistry.org/Part:BBa_R0040>BBa_R0040</a>).</p> <br />
<br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Fig.1 Genetic circuit for catechol degradation showing <i>XylE</i> biobricked under the TetR promoter|center]]<html><br />
<h3></h3><br />
<br />
<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of <i>XylE</i>.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Fig.2 Catechol 2,3-dioxygenase (<i>XylE</i>) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu <i>et al</i>., 1995.|center]]<html><br />
<br />
<p>The visual assays were performed with <i>E.coli</i> cells transformed with <a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a> as well as with <i>E.coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >BBa_K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part <a href=http://partsregistry.org/Part:BBa_K118021>BBa_K118021</a> was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was necessary to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011>BBa_K118011</a>). Catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the protocol written by the 2008 Edinburgh iGEM team.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.3 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added. |center]]<html><br />
<br />
<h2> Catechol into hydrocarbons? </h2><br />
<p>After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our xylE construct, we wondered if we could take this any further: perhaps converting it too into hydrocarbons. As catechol is the breakdown product of a number of different degradation pathways, this could be particularly useful. As 2-hydroxymuconate semialdehyde can be further metabolized to pyruvate and acetaldehyde, it seemed possible that these products could then be routed into the fatty acid biosynthesis pathway and converted to alkanes using the Petrobrick once again. Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct could be co-cultured with Petrobrick-containing cells to cooperatively metabolise catechol into hydrocarbons. </p><br />
<br />
<p> In orde to test this, we .... We tried this using both cells expressing the PetroBrick and __ cells with the oleT gene.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</html>}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/DecarboxylationTeam:Calgary/Project/OSCAR/Decarboxylation2012-10-03T22:36:53Z<p>MaggieRY: </p>
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<h2>Why Decarboxylation?</h2><br />
<br />
<p>Among the toxins found in the tailing ponds, naphthenic acids (NAs) are among the most harmful and the most common. Though there is great diversity within the NAs class of compounds, all share the common chemical feature of a carboxylic acid group. The carboxyl group is the primary cause for their toxicity, allowing these chemicals to traverse cell membranes <br />
and react with cellular materials (Frank et al, 2009). NAs are recalcitranct (not easily degraded), potentially harmful to the surrounding ecosystem (Clemente & Fedorak, 2005) and corrosive to extraction and transport equipment of petroleum materials (Slavcheva et al, 1999). Corrosion of pipelines leads to higher maintenance costs as well as the grim possibility of these and other toxins leaking into the environment. <br />
There is a need for methods to degrade NAs that are not prohibitively expensive or that would result in production of other hazardous chemicals.</p> <br />
<br />
<p>The main goal of OSCAR is to turn toxins like these into <br />
<br />
useable hydrocarbons by removing the carboxylic acid group(s) (Behar & Albrecht, 1984). <br />
<br />
Since NAs from petroleum deposits are a variable mixture, an enzymatic process with broad<br />
specificity is necessary. With the removal of the carboxylic acid <br />
<br />
moiety, we aim to produce alkanes suitable for use as fuel. The goal of this subproject was to find one or more suitable <br />
<br />
pathways to accomplish the decarboxylation of compounds such as <br />
<br />
NAs with the broadest specificity.</p><br />
<br />
<br />
<h2>The PetroBrick</h2><br />
<br />
<p>The 2011 Washington iGEM team developed the PetroBrick (<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?<br />
<br />
title=Part:BBa_K590025">BBa_K590025.</a>), a <br />
<br />
BioBrick consisting of two primary genes. These include acyl-ACP <br />
<br />
reductase (<i>AAR</i>), which reduces fatty acids bound to ACP to fatty <br />
<br />
aldehydes, and a second gene called aldehyde decarbonylase (<i>ADC</i>), <br />
<br />
which subsequently cleaves the entire aldehyde group and results <br />
<br />
in a hydrocarbon chain. Essentially this allows for hydrocarbons <br />
<br />
to be produced from glucose. What we realized though, is that <br />
<br />
the fatty acids that the PetroBrick targets, have a very <br />
<br />
similar structure to NAs.</p><br />
<br />
</html>[[File:UCalgary-Fatty-Acids-vs-NAs.jpg|500px|centre|thumb|Figure 1. A comparison of the structure of fatty acids and naphthenic acid]]<html><br />
<br />
<br />
<br />
<p>This lead us to believe that the PetroBrick may have the potential to turn NAs in to hydrocarbons and be a perfect solution to remediating NAs! First though, we needed to show that the PetroBrick did in fact work as expected. We had some difficulty with the DNA from the registry and had to request the constructs directly from the Washington team. Once we had the Petrobrick, we needed to verify that the Petrobrick would work in our hands as it did for the 2011 Washington team. <br />
<br />
Figures 2 and 3 demonstrate the function of the Petrobrick.</p><br />
</html>[[File:Calgary2012_PetrobrickVerificationGC.jpg|center|thumb|Figure 2: Gas Chromatograph demonstrating the differences in peak composition between an <i>E.coli</i> control and the Petrobrick. There was a large increase in a peak with a retention time of 12.25 min. suggesting that the Petrobrick was producing a new compound.|500px]]<html><br />
<br />
</html>[[File:Calgary2012_PetrobrickVerificationMS.jpg|center||thumb|Figure 3: Mass Spectra of the gas chromatograph peak at 12.25 min. The spectra suggests that the Petrobrick is selectively producing a C15 alkane. This is what was expected as determined by the Washington 2011 iGEM team.|500px]]<html><br />
<br />
<p>With the Petrobrick shown to be able to successfully produce alkanes, it was time to test it out on NAs, to see if <br />
they could be selectively converted into alkanes! This experiment used commercially available NAs fractions including a large number of different complex NAs compounds. </p><br />
<br />
<h2> Successful conversion of NA's into Hydrocarbons!</h2><br />
<br />
<br />
</html>[[File:Ucalgary_Decarboxylation_NaphthenicAcids_Results.png|center|thumb|Figure 4: The relative intensity of alkane production over a retention time in both <i>E.coli</i> that contain the PetroBrick, and in <i>E.coli</i> that are lacking the PetroBrick, as measured with GC-MS. NAs were used as a substrate. A NA standard was required to compare peaks.|700px]]<html><br />
<br />
</html>[[File:Ucalgary_Decarboxylation_Alkanes_Alkenes_Results.png|center|700px|thumb|Figure 5: The alkane and alkene mass spectrums generated by analysis of hydrocarbons produced from <i>E.coli</i> containing the PetroBrick as in Figure 2, using NAs as a substrate, as measured with GC-MS. Relative intensity to mass to charge ratio were compared.]]<html><br />
<br />
<p> The above graphs indicate that hydrocarbons were successfully produced from <i>E.coli</i> that contained the PetroBrick plasmid, as analysed with GC-MS. In Figure 2, <i>E.coli</i> containing the PetroBrick had significantly higher hydrocarbon peaks than in a control of <i>E.coli</i> that did not contain the PetroBrick plasmid. Not only was the PetroBrick able to degrade NAs into alkanes, but it was also able to produce alkenes as shown by Figure 3, indicating that the PetroBrick worked how we had expected it to! </p><br />
<br />
<br />
<h2><i>Nocardia</i> Carboxylic Acid Reductase (CAR)- Can we do better?</h2><br />
<br />
<p>Although we were successful using the Petrobrick to remove carboxyl groups from NAs, we wanted to improve on our results to see if we could get a higher yield or possibly target other compounds. One of our original fears in using the PetroBrick to <br />
decarboyxlate NAs was that the first enzyme AAR was reported to be highly specific for fatty acids bound to ACP. We had concerns about its compatibility with NAs and therefore sought another enzyme in the literature called carboxylic acid reductase (CAR) that was documented to perform a similar task as AAR, converting fatty acids to aldehydes, but with much lower specificity (He et al, 2004). This enzyme, from <i>N. iowensis</i> does not require covalent attachment to ACP so would <br />
likely be much broader in substrate specificity. It requires a second gene from <i>N. iowensis</i>, called Nocardia phosphopantetheinyl transferase (<i>NPT</i>) necessary to append a 4’- phosphopantetheine prosthetic group to CAR required for its full function (Venkitasubramanian et al, 2006).</p><br />
<br />
</html>[[File:Ucalgary Decarboxylation Team CAR Mechanism.jpg|center|350px|thumb|Figure 6. Mechanism of action of CAR]]<html><br />
<br />
<p>Another enzyme with the potential to remove carboxyl groups from NAs is olefin-forming fatty acid decarboxylase (<i>OleT</i>) from <i>Jeotgalicoccus sp. ATCC 8456</i>. This is a decarboxylase of the cytochrome P450 family that acts on fatty acids, but has also been documented to have low substrate specificity (Rude et al, 2011). What was attractive with this was that it was one single enzyme that go do the job of the PetroBrick! Now that we knew that our decarboxylation approach was valid, it was time to start testing and comparing this gene to the PetroBrick.</p><br />
<br />
<h2> Progress so far </h2><br />
<br />
<p> <i>CAR</i> and <i>NPT</i> were cloned from the host organism <i>N. iowensis</i> (NRRL 5646). CAR was ligated into the PET vector and verified by a restriction digest while <i>NPT</i> was cloned into pSB1C3(<a <br />
<br />
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902061">BBa_K902061.</a>) and similarly verified.</p><br />
<br />
<p><i>CAR</i> was cloned into pET47b+ (Novagen) plasmid due to six illegal cut sites(one XbaI site, two EcoRI sites, and three NotI <br />
<br />
sites) which made it unsuitable for the BioBrick construction vectors. We first attempted to use a multi-site <br />
<br />
mutagenesis derived from the QuikChange® Multi Site Directed Mutagenesis Kit, but this showed little success. Instead, a more <br />
<br />
time-consuming but effective series of conventional single-site <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">mutagenesis procedure</a> was favoured, using the Kappa Hi-Fi polymerase. The XbaI and EcoRI sites were eliminated <br />
<br />
first so that <i>CAR </i> can be moved from the pET Vector and ligated into the PSB1C3 vector (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902062">BBa_K902062.</a>). <i>OleT</i> was successfully amplified from the <i>Jeotgalicoccus sp. ATCC 8456</i>.<p> <br />
</p><br />
<p>Like <i>CAR</i>, <i>OleT</i> was inserted in a pET47b+ vector before placing it into a BioBrick vector, as two illegal cut sites adjacent to one another needed to be mutagenized. This part is now being ligated into pSB1C3. <br />
<br />
We are currently in the process of constructing all three parts under contorl of a <i>tetR </i> promoter and ribosomal binding site (<a href="http://partsregistry.org/Part:BBa_J13002">BBa_J13002</a>), and then constructing these composite parts together as outlined below.</p><br />
<br />
<h2>Final testing constructs</h2><br />
<br />
<p>Final testing constructs are almost complete. These are illustrated in figure 7 and will allow us to compare the three different approaches. Unfortunately, as Washington only sent us the PetroBrick and not the two individual components, we will have to compare a combination of the PetroBrick and CAR/NPT to the PetroBrick alone and to <i>OleT</i>. </p><br />
<br />
<p></html>[[File:Ucalgary_Decarboxylation_Team_J13002+car+J13002+npt+PetroBrick.png|centre|450px]]<html></html>[[File:Ucalgary Decarboxylation Team J13002+oleT.png|centre|350px|thumb|Figure 7. Final constructs required for validating and comparing different decarboxylation approaches]]<html></p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-03T20:07:52Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
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<br />
<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement, many toxic compounds are produced. These have become a huge problem in our society resulting in land, water, and air contamination. The toxins consist of a variety different types of compounds. Air contaminants consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to a variety of environmental issues including green house gas accumulation and acid rain. Similarly water contaminants often consist of complex mixtures of compounds including highly toxic phenols and aromatic compounds, corrosive and additionally toxic carboxylic acid containing compounds, sulfur, and nitrogen containing compounds. These often have complex structures and cause acute toxicity to wild life. Classical examples of water contamination include tailing ponds produced from the oil extraction process. Finally, land areas can become contaminated as a result of these toxic compounds leaching into ground water sources, spills or accidental release of waste products into the environment, and other ways. </p><br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>Presently, there are a variety of solutions to removing these compounds from the environment. This has been facilitated by stricter regulations from government bodies as well as better chemical and physical techniques for reducing the release of these compounds. Toxins which are released into the environment can be removed using various chemical agents or by physically removing contaminated soil or water areas and storing these products in contained areas. However there is still no efficient, environmentally healthy mechanism for this to occur. <b>What needs to occur in order to better remediate these toxin's from the environment?</b> We require a method to be able to easily detect where toxins are and also have a system for remediating them. Microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transform them into other products. Therefore we can harness these abilities to apply synthetic biology to these problems.</p><br />
<br><br />
<p><b><center>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</center></b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
<br />
<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED or the Functional Robust Electrochemical Detector, is capable of detecting various toxic components through an electrochemical response at the same time. We illustrated how this sensor could work by showing that it has the potential to detect toxins in contaminated water. Additionally, we developed a minaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
</p><br />
<p><br />
OSCAR or the Optimized System for Carboxylic Acid Remediation is designed specifically to target naphthenic acids (NAs) the carboxylic acid containing compounds in the tailing ponds for their degradation. Using the Petrobrick (<b>BBa______</b>) we were able to convert various different naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the petrobrick. <b>FILL THIS IN DEPENDENT ON CATECHOL ASSAY DATA TONIGHT!</b><br />
<br />
</p><br />
</html><br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-03T19:34:14Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Catechol Degradation|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<br />
<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006, Schweigert <i>et al.</i>, 2001)). The chemical properties of catechol allow it to react with biomolecules, to cause serious cellular damage including DNA breakage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). </p><br />
<p><br />
Catechol is characterized as having a benzene ring with two hydroxyl groups at the 2,3 position. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983). This product can then be further metabolized to pyruvate and acetaldehyde; products which can then be routed into the fatty acid biosynthesis pathway and converted to alkanes with the Petrobrick.</p><br />
<br />
<p><br />
The current iGEM Part repository has two BioBricks available of <i>xylE</i>. One contained <i>xylE</i> with its native ribosome-binding site (part: <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>), while the other part contained <i>xylE</i> under the glucose-repressible promoter cstA (Part: <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>xylE</i> repressed by using the TetR promoter (Part:<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>).</p> <br />
<br />
<br />
<br />
<br />
<h3></h3><br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Fig.1 Genetic circuit for catechol degradation showing <i>XylE</i> biobricked under the TetR promoter|center]]<html><br />
<h3></h3><br />
<br />
<br />
<br />
<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of <i>XylE</i>.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Fig.2 Catechol 2,3-dioxygenase (<i>XlyE</i>) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu ''et al''., 1995.|center]]<html><br />
<br />
<p>The visual assays were performed with <i>E.coli</i> cells transformed with <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> as well as with <i>E.coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was necessary to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011 >K118011</a>). Catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the protocol written by the 2008 Edinburgh iGEM team.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.3 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added. |center]]<html><br />
<br />
<p><br />
Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct are co-cultured with Petrobrick containing cells to cooperatively metabolise catechol into hydrocarbons. </p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</html>}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-03T19:29:25Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Catechol Degradation|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<br />
<br />
<p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006, Schweigert <i>et al.</i>, 2001)). The chemical properties of catechol allow it to react with biomolecules, to cause serious cellular damage including DNA breakage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). </p><br />
<p><br />
Catechol is characterized as having a benzene ring with two hydroxyl groups at the 2,3 position. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983). This product can then be further metabolized to pyruvate and acetaldehyde; products which can then be routed into the fatty acid biosynthesis pathway and converted to alkanes with the Petrobrick.</p><br />
<br />
<p><br />
The current iGEM Part repository has two BioBricks available of <i>xylE</i>. One contained <i>xylE</i> with its native ribosome-binding site (part: <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>), while the other part contained <i>xylE</i> under the glucose-repressible promoter cstA (Part: <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>xylE</i> repressed by using the TetR promoter (Part:<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>).</p> <br />
<br />
<br />
<br />
<br />
<h3></h3><br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Fig.1 Genetic circuit for catechol degradation showing <i>XylE</i> biobricked under the TetR promoter|center]]<html><br />
<h3></h3><br />
<br />
<br />
<br />
<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of <i>XylE</i>.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Fig.2 Catechol 2,3-dioxygenase (<i>XlyE</i>) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu ''et al''., 1995.|center]]<html><br />
<br />
<p>The visual assays were performed with <i>E.coli</i> cells transformed with <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> as well as with <i>E.coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was done to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011 >K118011</a>). The catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the previous assay done by the 2008 Edinburgh iGEM team.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.3 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added. |center]]<html><br />
<br />
<p><br />
Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct are co-cultured with Petrobrick containing cells to cooperatively metabolise catechol into hydrocarbons. </p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</html>}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradationTeam:Calgary/Project/OSCAR/CatecholDegradation2012-10-03T19:18:29Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Catechol Degradation|<br />
<br />
CONTENT=<html><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_OSCAR_Catechol_Low-Res.png" style="float: right; padding: 10px;"></img><br />
<p><b>****This section needs work. Why are we degrading catechol? What part did we use? What is the number?</b></p><br />
<br />
<p>Catechol is another toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt <i>et al.</i>, 2006, Schweigert <i>et al.</i>, 2001)). The chemical properties of catechol allow it to react with biomolecules like DNA, proteins and membranes (Schweigert <i>et al.</i>, 2001). These interactions can cause serious damage including DNA breakage, enzyme inactivation and membrane uncoupling (Schweigert <i>et al.</i>, 2001). </p><br />
<p><br />
Catechol is characterized as having a benzene ring with two hydroxyl groups emanating from adjacent carbons. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983). This product can then be further metabolized to pyruvate and acetaldehyde; products which can then be routed into the fatty acid biosynthesis pathway and converted to alkanes with the Petrobrick.</p><br />
<br />
<p><br />
The current iGEM Part repository has two BioBricks available of <i>xylE</i>. One contained <i>xylE</i> with its native ribosome-binding site (part: <a href=http://partsregistry.org/Part:BBa_J33204>J33204</a>), while the other part contained <i>xylE</i> under the glucose-repressible promoter cstA (Part: <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a>). Given that <i>E. coli</i> is grown in the presence of glucose, we designed a new construct to keep <i>xylE</i> repressed by using the TetR promoter (Part:<a href= http://partsregistry.org/Part:BBa_R0040>R0040</a>).</p> <br />
<br />
<br />
<br />
<br />
<h3></h3><br />
</html>[[File:UCalgary2010_R0040-XylE.png|400px|thumb|Fig.1 Genetic circuit for catechol degradation showing <i>XylE</i> biobricked under the TetR promoter|center]]<html><br />
<h3></h3><br />
<br />
<br />
<br />
<p>Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of <i>XylE</i>.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_to_2-HMS.PNG|400px|thumb|Fig.2 Catechol 2,3-dioxygenase (<i>XlyE</i>) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu ''et al''., 1995.|center]]<html><br />
<br />
<p>The visual assays were performed with <i>E.coli</i> cells transformed with <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> as well as with <i>E.coli</i> cells transformed with the newly constructed part (<a href=http://partsregistry.org/Part:BBa_K902048 >K902048</a>) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part <a href=http://partsregistry.org/Part:BBa_K118021>K118021</a> was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was done to avoid the glucose in the LB from repressing the cstA promoter (<a href=http://partsregistry.org/Part:BBa_K118011 >K118011</a>). The catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the previous assay done by the 2008 Edinburgh iGEM team.</p><br />
<br />
</html>[[File:UCalgary2012_Catechol_assay.jpg|500px|thumb|Fig.3 Results of the catechol visual assay using the part K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM for varying times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). After this incubation in M9-MM the cells were spun down and catechol was added to the supernatant to bring it to a concentration of 0.1 M. The amount of time didn't affect the colour change in the cultures containing the <i>XylE</i> gene. The right most tube was a culture of <i>E.coli</i> cells without the <i>XylE</i> gene that was used as a control. The controls supernatant remained clear when the catechol was added. |center]]<html><br />
<br />
<p><br />
Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct are co-cultured with Petrobrick containing cells to cooperatively metabolise catechol into hydrocarbons. </p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</html>}}</div>MaggieRY