http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Myarcell&year=&month=2012.igem.org - User contributions [en]2024-03-29T06:05:39ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:47:38Z<p>Myarcell: </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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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
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<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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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><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 />
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<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 />
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<h2>Have we accomplished our goal?</h2><br />
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<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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</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 />
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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. All our electrochemical protocols can be found here. The results of this assay can be shown below.</p><br />
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</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 />
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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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}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T03:25:53Z<p>Myarcell: </p>
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<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
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<h2> <u>Initial Interviews</u> </h2><br />
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<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 was that engineered organisms might outcompete existing tailings ponds bacteria, and Christine had similar reservations. 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. There is research using other systems such as algal bioremediation, but practical implementations of biology in the oil sands are rare. 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 />
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<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<p>We were also able to consult with BioAlberta president Ryan Radke on the viability of using biologically based systems in the petroleum industry. He believed that bioremediation technologies will be critical to the future of the oil sands in the next 10-15 years, however he raised the concern of how we could sell biotechnology to an industry that is already very successful from a profit point of view. He emphasized the social welfare benefits that the industry, under heavy environmental criticism, could gain from this technology as a major selling point and stressed the need to educate the oil industry about the benefits of synthetic biology. This would involve putting aspects of our system into large-scale, comprehensive terms to sell to those without a biology background. For example, quantifying the amount of toxins that the system can process per unit time and the value of hydrocarbons produced is something that could potentially appeal to investors. We feel as though the synthetic biology <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">dialogue with the Oil Sands Leadership Initiative</a> was a great start to bridging the gap between the petroleum industry and synthetic biology. From a public perspective point of view we discussed the need to stress the fact our system is simply a modification of "natural" processes - that is we are only modifying bacteria native to the tailings ponds, not introducing new organisms to the environment. To further address safety concerns, we also need to emphasize the multiple layers of <a href="https://2012.igem.org/Team:Calgary/Safety">biological and physical control</a> that we plan to design.</p> <br />
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<h2><u>Follow-Up Interviews</u></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 investigate ways of dealing with the clay and silt particles in tailings pond water that would enter our bioreactor system. This can be a major problem since these 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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our present design were issues with scale-up. These were things such as the amount of toxins that would have to be processed to provide constant generation of product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do toward this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we should look at addressing the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to these fine tailings and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
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<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that Mr. Sawchuk brought up was that there needs to be extra safety features put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
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<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and how much a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we have been experimenting with getting our bioreactor to work and have performed an initial validation assay to demonstrate that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! Calculating exact costs is a tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
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</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">OSLI</a> is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Scale up</a> of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Zvonko Burkus from Alberta Environment Discusses the Project in Detail</h3><br />
<p>Zvonko is a process water and policy specialist for Alberta Environment. Zvonko was happy to discuss with us at length about this project. Notably, there were concerns about the use of both FRED and OSCAR in active tailings ponds, since naphthenic acids are known surfactants which help with bitumen detachment from the sand particles. FRED was seen as something which could be more useful in a live-monitoring system, since currently there are no such systems for organic compounds, which is a possible direction for us.</p><br />
<p>We were warned that the oil industry is rather traditional, but as we have seen from our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaboration">OSLI dialogue</a>, it appears that more companies are beginning to see the potential benefit of biology in solving various issues with the oil sands.</p><br />
<p>Some interesting points for us to explore further included examining whether or not the intermediates of our metabolic pathways were more or less toxic than the starting compounds, and expanding our scope to target other compounds such as polyaromatic hydrocarbons, chlorides, composite tailings, polyphenols, and hydrogen sulfide.</p><br />
<p>This interview was a phone recording. Please download it here: </p><br />
<br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T03:21:17Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 was that engineered organisms might outcompete existing tailings ponds bacteria, and Christine had similar reservations. 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. There is research using other systems such as algal bioremediation, but practical implementations of biology in the oil sands are rare. 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 />
</div><br />
<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<p>We were also able to consult with BioAlberta president Ryan Radke on the viability of using biologically based systems in the petroleum industry. He believed that bioremediation technologies will be critical to the future of the oil sands in the next 10-15 years, however he raised the concern of how we could sell biotechnology to an industry that is already very successful from a profit point of view. He emphasized the social welfare benefits that the industry, under heavy environmental criticism, could gain from this technology as a major selling point and stressed the need to educate the oil industry about the benefits of synthetic biology. This would involve putting aspects of our system into large-scale, comprehensive terms to sell to those without a biology background. For example, quantifying the amount of toxins that the system can process per unit time and the value of hydrocarbons produced is something that could potentially appeal to investors. We feel as though the synthetic biology <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">dialogue with the Oil Sands Leadership Initiative</a> was a great start to bridging the gap between the petroleum industry and synthetic biology. From a public perspective point of view we discussed the need to stress the fact our system is simply a modification of "natural" processes - that is we are only modifying bacteria native to the tailings ponds, not introducing new organisms to the environment. To further address safety concerns, we also need to emphasize the multiple layers of <a href="https://2012.igem.org/Team:Calgary/Safety">biological and physical control</a> that we plan to design.</p> <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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 investigate ways of dealing with the clay and silt particles in tailings pond water that would enter our bioreactor system. This can be a major problem since these 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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our present design were issues with scale-up. These were things such as the amount of toxins that would have to be processed to provide constant generation of product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do toward this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we should look at addressing the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to these fine tailings and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that Mr. Sawchuk brought up was that there needs to be extra safety features put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and how much a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we have been experimenting with getting our bioreactor to work and have performed an initial validation assay to demonstrate that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">OSLI</a> is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Scale up</a> of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Zvonko Burkus from Alberta Environment Discusses the Project in Detail</h3><br />
<p>Zvonko is a process water and policy specialist for Alberta Environment. Zvonko was happy to discuss with us at length about this project. Notably, there were concerns about the use of both FRED and OSCAR in active tailings ponds, since naphthenic acids are known surfactants which help with bitumen detachment from the sand particles. FRED was seen as something which could be more useful in a live-monitoring system, since currently there are no such systems for organic compounds, which is a possible direction for us.</p><br />
<p>We were warned that the oil industry is rather traditional, but as we have seen from our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaboration">OSLI dialogue</a>, it appears that more companies are beginning to see the potential benefit of biology in solving various issues with the oil sands.</p><br />
<p>Some interesting points for us to explore further included examining whether or not the intermediates of our metabolic pathways were more or less toxic than the starting compounds, and expanding our scope to target other compounds such as polyaromatic hydrocarbons, chlorides, composite tailings, polyphenols, and hydrogen sulfide.</p><br />
<p>This interview was a phone recording. Please download it here: </p><br />
<br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T03:16:58Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 was that engineered organisms might outcompete existing tailings ponds bacteria, and Christine had similar reservations. 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. There is research using other systems such as algal bioremediation, but practical implementations of biology in the oil sands are rare. 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 />
</div><br />
<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<p>We were also able to consult with BioAlberta president Ryan Radke on the viability of using biologically based systems in the petroleum industry. He believed that bioremediation technologies will be critical to the future of the oil sands in the next 10-15 years, however he raised the concern of how we could sell biotechnology to an industry that is already very successful from a profit point of view. He emphasized the social welfare benefits that the industry, under heavy environmental criticism, could gain from this technology as a major selling point and stressed the need to educate the oil industry about the benefits of synthetic biology. This would involve putting aspects of our system into large-scale, comprehensive terms to sell to those without a biology background. For example, quantifying the amount of toxins that the system can process per unit time and the value of hydrocarbons produced is something that could potentially appeal to investors. We feel as though the synthetic biology <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">dialogue with the Oil Sands Leadership Initiative</a> was a great start to bridging the gap between the petroleum industry and synthetic biology. From a public perspective point of view we discussed the need to stress the fact our system is simply a modification of "natural" processes - that is we are only modifying bacteria native to the tailings ponds, not introducing new organisms to the environment. To further address safety concerns, we also need to emphasize the multiple layers of <a href="https://2012.igem.org/Team:Calgary/Safety">biological and physical control</a> that we plan to design.</p> <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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 investigate ways of dealing with the clay and silt particles in tailings pond water that would enter our bioreactor system. This can be a major problem since these 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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our present design were issues with scale-up. These were things such as the amount of toxins that would have to be processed to provide constant generation of product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do toward this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we should look at addressing the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to these fine tailings and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">OSLI</a> is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Scale up</a> of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Zvonko Burkus from Alberta Environment Discusses the Project in Detail</h3><br />
<p>Zvonko is a process water and policy specialist for Alberta Environment. Zvonko was happy to discuss with us at length about this project. Notably, there were concerns about the use of both FRED and OSCAR in active tailings ponds, since naphthenic acids are known surfactants which help with bitumen detachment from the sand particles. FRED was seen as something which could be more useful in a live-monitoring system, since currently there are no such systems for organic compounds, which is a possible direction for us.</p><br />
<p>We were warned that the oil industry is rather traditional, but as we have seen from our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaboration">OSLI dialogue</a>, it appears that more companies are beginning to see the potential benefit of biology in solving various issues with the oil sands.</p><br />
<p>Some interesting points for us to explore further included examining whether or not the intermediates of our metabolic pathways were more or less toxic than the starting compounds, and expanding our scope to target other compounds such as polyaromatic hydrocarbons, chlorides, composite tailings, polyphenols, and hydrogen sulfide.</p><br />
<p>This interview was a phone recording. Please download it here: </p><br />
<br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-27T03:00:55Z<p>Myarcell: </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 disrupting metabolism or the limitations of 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 its 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 its 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>Myarcellhttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-27T02:58:18Z<p>Myarcell: </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 different 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. 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 can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a 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>Pseudomonas spp. </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 <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> 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 </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence 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 />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><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 2: 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 </i> PF-5 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 <i>et al</i>., 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 />
<h3>Mutant Strain Characterization</h3><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 />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and 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 />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><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 3: Transposons: 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 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. 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 5: 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 6: 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 7: 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 />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8: Stress response screen on <i>P. fluorescens</i> Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. </p><p> <br />
Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. The results of this can be seen on the Synergy page.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/FRED/DetectingTeam:Calgary/Project/FRED/Detecting2012-10-27T02:55:57Z<p>Myarcell: </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 different 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. 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 can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a 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>Pseudomonas spp. </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 <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> 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 </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence 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 />
<a name="library"></a><h2>Method Design</h2><br />
<h3>Mutant Library Generation</h3><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 2: 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 </i> PF-5 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 <i>et al</i>., 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 />
<h3>Mutant Strain Characterization</h3><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 />
<h3>Self-Cloning and Sequencing</h3><br />
<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and 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 />
<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3><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 3: Transposons: 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 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. 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 5: 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 6: 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 7: 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 />
<p align="justify"><br />
From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p><br />
<p align="justify"><br />
</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8: Stress response screen on <i>P. fluorescens</i> Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p><br />
<p><br />
These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p><br />
<br />
<h2>Promoter Constructs Isolated</h2><br />
<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. </p><p> <br />
Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. <b>The results of this can be seen on the Synergy page</b>.</p><br />
<br><br />
<br />
<br />
<br />
<br />
<br />
</p><br />
<br />
</html><br />
<br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T02:42:19Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 was that engineered organisms might outcompete existing tailings ponds bacteria, and Christine had similar reservations. 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. There is research using other systems such as algal bioremediation, but practical implementations of biology in the oil sands are rare. 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 />
</div><br />
<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<p>We were also able to consult with BioAlberta president Ryan Radke on the viability of using biologically based systems in the petroleum industry. He believed that bioremediation technologies will be critical to the future of the oil sands in the next 10-15 years, however he raised the concern of how we could sell biotechnology to an industry that is already very successful from a profit point of view. He emphasized the social welfare benefits that the industry, under heavy environmental criticism, could gain from this technology as a major selling point and stressed the need to educate the oil industry about the benefits of synthetic biology. This would involve putting aspects of our system into large-scale, comprehensive terms to sell to those without a biology background. For example, quantifying the amount of toxins that the system can process per unit time and the value of hydrocarbons produced is something that could potentially appeal to investors. We feel as though the synthetic biology <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">dialogue with the Oil Sands Leadership Initiative</a> was a great start to bridging the gap between the petroleum industry and synthetic biology. From a public perspective point of view we discussed the need to stress the fact our system is simply a modification of "natural" processes - that is we are only modifying bacteria native to the tailings ponds, not introducing new organisms to the environment. To further address safety concerns, we also need to emphasize the multiple layers of biological and physical control that we plan to design.</p> <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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 investigate ways of dealing with the clay and silt particles in tailings pond water that would enter our bioreactor system. This can be a major problem since these 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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our present design were issues with scale-up. These were things such as the amount of toxins that would have to be processed need to provide constant generation of product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T02:18:11Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 was that engineered organisms might outcompete existing tailings ponds bacteria, and Christine had similar reservations. 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. There is research using other systems such as algal bioremediation, but practical implementations of biology in the oil sands are rare. 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 />
</div><br />
<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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T02:17:18Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 was that engineered organisms might outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had similar reservations. 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. There is research using other systems such as algal bioremediation, but practical implementations of biology in the oil sands are rare. 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 />
</div><br />
<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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T02:12:16Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 was that engineered organisms might outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had similar reservations. 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 />
</div><br />
<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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T02:11:33Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 was that engineered organisms might outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar reservation. 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 />
</div><br />
<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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T02:07:13Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 engineered organisms might outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar reservation. 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 />
</div><br />
<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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T02:04:19Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 raise concerns among legislators and industrial leaders. <br />
</p><br />
<br />
<h2> <u>Initial Interviews</u> </h2><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 />
</div><br />
<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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T01:56:19Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team undertook 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 />
<h2> <u>Initial Interviews</u> </h2><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 />
</div><br />
<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 />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></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 />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design"> structural and genetic killswitch devices</a>. In the later part of our project, we have also been trying to work on establishing a <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">glycine auxotrophic killswitch</a> to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<p>Gordon Lambert is the VP Sustainable Development at Suncor Energy Inc. We asked him whether or not the oil sands industry would find technology such as this useful. There was a very positive response. The Oil Sands Leadership Initiative is very keen on searching for any solutions to tackle the tailings ponds, which are considered to be one of the biggest issues in the oil sands currently. OSLI is collaborating with organizations that run competitions globally for oil sands solutions and other bodies such the Canada's Oil Sands Innovation Alliance (COSIA). Similar to Kelly Roberge's comment, mature fine tailings can be dried and solidified, but in turn it liberates water from the clay and sand. This water cannot be used for any industrial purposes until it is detoxified. Ideally, this water can be detoxified sufficiently to be returned as tailings pond surface water and become reusable in the bitumen extraction process.</p><br />
<p>In order to deploy our biosensor and bioreactor system, it was suggested that we look into various regulatory boards within Alberta such as Alberta Environment and the Energy Resources Conservation Board (ERCB) to attempt to obtain permits to begin attempting pilot programs. Scale-up of the bioreactor is also a major consideration in order for us to push it off the bench and into the field.</p><br />
<p>The full interview can be found below. If it does not load, <a href="http://www.youtube.com/watch?v=7KbEjQVUsFA">click here</a>.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:51:49Z<p>Myarcell: </p>
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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. These compounds have an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 of the potentiostat for the prototype, validated this device in the wetlab, and designed our own software, which we have made available to everyone. <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 containing heterocyclic compounds. 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 in 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! Our meeting with the Oilsands Leadership Initiative (OSLI) has affirmed 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 emphasized that while the concept sounds great, it is 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 toward 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 from the outset, both FRED and OSCAR were designed in a safe and functional way. This involved incorporating physical containment features in the biosensor and bioreactor designs and employing biological kill switch mechanisms.</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 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 had its debut at the centre and educated adults and kids on synthetic biology in a fun way! </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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:39:16Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
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<head><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. These compounds have an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 of the potentiostat for the prototype, validated this device in the wetlab, and designed our own software, which we have made available to everyone. <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 containing heterocyclic compounds. 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 in 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! Our meeting with the Oilsands Leadership Initiative (OSLI) has affirmed 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 emphasized that while the concept sounds great, it is 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:37:50Z<p>Myarcell: </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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float:left;<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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 of the potentiostat for the prototype, validated this device in the wetlab, and designed our own software, which we have made available to everyone. <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 containing heterocyclic compounds. 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 in 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! Our meeting with the Oilsands Leadership Initiative (OSLI) has affirmed 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, it is 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:36:10Z<p>Myarcell: </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 />
margin-left: 30px;<br />
float:left;<br />
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background: #5BB5E8;<br />
margin-left: 4px;<br />
float:left;<br />
}<br />
#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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 of the potentiostat for the prototype, validated this device in the wetlab, and designed our own software, which we have made available to everyone. <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 containing heterocyclic compounds. 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 in 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! Our meeting with the Oilsands Leadership Initiative (OSLI) has affirmed 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:33:10Z<p>Myarcell: </p>
<hr />
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TITLE=Project Overview|CONTENT=<br />
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<body><br />
<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. These compounds have an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 of the potentiostat for the prototype, validated this device in the wetlab, and designed our own software, which we have made available to everyone. <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 containing heterocyclic compounds. 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 in 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:27:53Z<p>Myarcell: </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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float:left;<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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 of the potentiostat for the prototype, validated this device in the wetlab, and designed our own software, which we have made available to everyone. <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 containing heterocyclic compounds. 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:26:51Z<p>Myarcell: </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 />
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#oscarbox:hover{<br />
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</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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 of the potentiostat for the prototype, validated this device in the wetlab, and designed software, which we have made available to everyone. <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 containing heterocyclic compounds. 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:20:54Z<p>Myarcell: </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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 thus becomes 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing heterocyclic compounds. 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:19:02Z<p>Myarcell: </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 />
margin-left: 30px;<br />
float:left;<br />
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#fredbox:hover{<br />
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}<br />
#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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing heterocyclic compounds. 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:17:52Z<p>Myarcell: </p>
<hr />
<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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing heterocyclic compounds. 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 />
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}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T01:14:57Z<p>Myarcell: </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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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 is designed to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and to convert these toxic components into usable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing 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>Myarcellhttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T01:02:49Z<p>Myarcell: </p>
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</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magnesium riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. In addition, one of our two 'toxin-sensing' transposon hits was characterized electrochemically, demonstrating its ability to respond to and report on NAs at levels detectable by our electrochemical reporting system. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> and <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> pages respectively. </p></li><br />
<br />
<li><p> Performed an actual test of our biosensor using tailings pond water, showing that we can detect toxins found in tailings. In addition, performed an actual "field test" of our prototype to demonstrate its feasibility and ease of use outside a laboratory setting. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. In addition, we performed an actual validation assay of our bioreactor, showing that we can use it to grow hydrocarbon producing cells and use our belt skimming device to harvest the hydrocarbons. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<li><p>Performed initial assays on a glycine knockout strain of <i>E. coli</i>, characterizing its survival in differing concentrations of glycine, its ability to work in conjunction with one of our inducible killswitch constructs (<a href=blue href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>) and finally its ability to work with the Petrobrick, actually substantially increasing our out put of hydrocarbons when grown with glycine as compared to a <i>DH5alpha</i> strain. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
<br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T00:56:23Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
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</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magnesium riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. In addition, one of our two 'toxin-sensing' transposon hits was characterized electrochemically, demonstrating its ability to respond to and report on NAs at levels detectable by our electrochemical reporting system. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> and <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> pages respectively. </p></li><br />
<br />
<li><p> Performed an actual test of our biosensor using tailings pond water, showing that we can detect toxins found in tailings. In addition, performed an actual "field test" of our prototype to demonstrate its feasibility and ease of use outside a laboratory setting. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. In addition, we performed an actual validation assay of our bioeacotr, showing that we can use it to grow hydrocarbon producing cells and use our belt skimming device to harvest the hydrocarbons. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<li><p>Performed initial assays on a glycine knockout strain of <i>E. coli</i>, characterizing its survival in differing concentrations of glycine, its ability to work in conjunction with one of our inducible killswitch constructs (<a href=blue href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>) and finally its ability to work with the Petrobrick, actually substantially increasing our out put of hydrocarbons when grown with glycine as compared to a <i>DH5alpha</i> strain. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
<br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
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</html><br />
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}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T00:50:38Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
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</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magnesium riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. In addition, one of our two 'toxin-sensing' transposon hits was characterized electrochemically, demonstrating its ability to respond to and report on NAs at levels detectable by our electrochemical reporting system. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> and <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> pages respectively. </p></li><br />
<br />
<li><p> Performed an actual test of our biosensor using tailings pond water, showing that we can detect toxins found in tailings. In addition, performed an actual "field test" of our prototype to demonstrate its feasibility and ease of use outside a laboratory setting. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. In addition, we performed an actual validation assay of our bioeacotr, showing that we can use it to grow hydrocarbon producing cells and use our belt skimming device to harvest the hydrocarbons. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<li><p>Performed initial assays on a glycine knockout strain of <i>E. coli</i>, characterizing its survival in differing concentrations of glycine, its ability to work in conjunction with one of our inducible killswitch constructs (<a href=blue href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>) and finally its ability to work with the Petrobrick, actually substantially increasing our out put of hydrocarbons when grown with glycine as compared to a <i>DH5alpha</i> strain. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
<br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
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</html><br />
<br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T00:28:25Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
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<body><br />
<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. These compounds have an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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 increasingly important, 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 by 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. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T00:24:54Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
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<style><br />
#fredbox{<br />
width: 320px;<br />
height: 215px;<br />
background: #58CD45;<br />
margin-left: 30px;<br />
float:left;<br />
}<br />
#fredbox:hover{<br />
background: #94FF7D;<br />
}<br />
#oscarbox{<br />
width: 320px;<br />
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}<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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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 kept contained in the ponds. This creates a susceptibility toward 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. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T00:16:04Z<p>Myarcell: </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 an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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, which causes 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. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing 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>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-27T00:10:46Z<p>Myarcell: </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 />
margin-left: 30px;<br />
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float:left;<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. These compounds have an enormous environmental impact, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants 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. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. 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 containing 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>Myarcellhttp://2012.igem.org/Team:Calgary/Project/AccomplishTeam:Calgary/Project/Accomplish2012-10-26T23:53:57Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Accomplishments|<br />
CONTENT=<br />
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<head><br />
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<br />
<h2> Our team had many accomplishments throughout the summer of 2012!</h2><br />
<p>Please see the <b>Post Regional</b> page for our results between Regionals and the Finals!<br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 250px; padding: 10px;"></img><br />
<ul><li><p> Established a <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">dialogue between industry experts</a> in order to inform the design of our project.</p></li><br />
<li><p>Led a discussion through the oil sands leadership initiative (OSLI) on the need and potential usefulness of <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations#initiative"><br />
synthetic biology in the oil sands</a>.</p></li><br />
<li><p>Submitted <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">novel riboswitch, promoter and regulatory parts</a> for use in the tight control of killswitch applications and beyond.</p></li><br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation#killswitch">Submitted and characterized</a> both a magnesium riboswitch/promoter GFP construct and a magnesium riboswitch/promoter kill gene construct.</p></li><br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization#UBC">Partnered with the UBC iGEM team</a> in order to build and better optimize the <i>dsz</i> desulfurization operon.</p></li><br />
<li><p>Showcased our project to our city and the world through various outreach initiatives including a <a class="orange" href="https://2012.igem.org/Team:Calgary/Outreach/TEDxCalgary">TEDxCalgary City 2.0 talk</a>.</p></li><br />
<li><p>Premiered and beta-tested our <a class="orange" href="https://2012.igem.org/Team:Calgary/Outreach/VideoGame">video game LAB ESCAPE!</a> at the <a class="orange" href="http://www.sparkscience.ca">Calgary Telus Spark World of Science</a>.</p></li></ul><br />
<br />
<br><br />
<br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<img src="https://static.igem.org/mediawiki/2012/3/31/UCalgary2012_FRED_Index_Box.png" style="float: right; padding: 10px;"></img><br />
<ul><br />
<li><p> Constructed a <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting#library">transposon library</a> in <i>Pseudomonas</i>, identifying two positive hits sensitive to a variety of tailings pond toxins.</p></li><br />
<li><p>Submitted and electrochemically characterized the function of <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting#hydrolase">two novel hydrolase enzymes</a> from <i>E. coli</i>, demonstrating the validity and potential of a <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting#output">triple-output system</a> with high sensitivity and little background noise.</p></li><br />
<li><p>Designed and wet-lab verified a <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">kinetic model</a> of electrochemical gene expression.</p></li><br />
<li><P>Designed both <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype#hardware">hardware</a> and <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype#software">software</a> for a biosensor prototype.</p></li></ul><br />
<br />
<br />
<br><br />
<br />
<p><b>In terms of <FONT COLOR=#1088CC>OCSAR</FONT>, we...</b></p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_OSCAR_Index_Box.png" style="float: right; padding: 10px;"></img><br />
<ul><br />
<br />
<li><p>Demonstrated the successful conversion of <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation#Petrobrick"><br />
naphthenic acids into hydrocarbons</a> using Washington 2011's PetroBrick.</p></li><br />
<br />
<li><P>Documented the functionality of the enzyme <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation#OleT">OleT</a>, an alternative enzyme to the PetroBrick for producing <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation#TestingOleT"><br />
alkenes from fatty acids</a>.</p></li><br />
<li><P>Modified an <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">existing <i>xylE</i></a> part to show the degradation of <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation#Catechol">catechol into a product, which is then degraded into hydrocarbons</a> using the PetroBrick or OleT.</p></li><br />
<li><P>Designed, built, and tested a <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">functioning bioreactor</a> system in which to house our toxin degrading strain.</p></li><br />
<li><P>Used flux variability analysis to <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis#Flux"><br />
optimize the production of our hydrocarbons</a>, surpassing Washington’s previous results through modification of growth media.</p></li><br />
<li><P>Demonstrated the successful <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization#Degradation">degradation of carbazole and DBT</a> by our model strains.</p></li><br />
<li><P>Submitted sequenced BioBricks for the removal of <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation#nitrogen">nitrogen</a> and <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization#sulfur"><br />
sulfur</a> from various compounds and mutagenized eight separate genes to remove illegal cut sites.</p></li><br />
<li><P>Submitted and characterized a new <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization#catalase">catalase generator</a> as well as a <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization#catalase">novel oxido-reductase enzyme</a> for use in our desulfurization project.</p></li><br />
<li><P><b><FONT COLOR=#1088CC>Had an amazing summer and learned a ton!</FONT></b></p></li></ul><br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/DesignTeam:Calgary/Project/HumanPractices/Design2012-10-26T23:36:37Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Preliminary Design Considerations|<br />
CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 250px;"></img><br />
<br />
<p>FRED and OSCAR have been tasked with jobs that require them to be outside of a laboratory environment. Our discussions with industry experts emphasized the need to design a system that minimized the chance of bacteria escaping into the environment. Despite our belief that due to the increased metabolic load FRED and OSCAR are undertaking they would not be able to outcompete any native bacteria, we took these concerns to heart when we designed our project. We have designed multiple layers of controls for each system, utilizing both biological and physical controls.</p><br />
<br />
<h2>Physical: The first line of defense</h2><br />
<br />
<p>The best way to prevent FRED and OSCAR from spreading into the environment is to make sure that they cannot get to it. As such both our bioreactor and biosensor prototypes involve isolating the bacteria in closed systems. In our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">biosensor</a> we seal the tubes with a one way valve with FRED trapped inside. The tailings sample is added through the one way valve and then when the testing is done the cap is twisted slightly to release bleach into the sealed system. After the bleach is added the tube is disposed of in a safe manner.</p><br />
<br />
<p>OSCAR presents more of a challenge, as he needs to remain in one place for an extended period of time to perform his tasks. For this we have created a <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> house for him. In this sealed system filtered air is bubbled in to keep oxygen levels optimal while a HEPA filter is used to screen air coming out. To extract any hydrocarbons from the reactor a belt skimmer is used that selectively picks up oil while leaving bacteria behind. When the oil is separated from the belt skimmer it is exposed to UV to kill any bacteria, and then is placed into a fractional distillator that heats to 400&deg;C to separate the hydrocarbons from each other.</p><br />
<br />
<h2>Biological: Preparing for the worst</h2><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor to favor adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been criticized in previous iGEM systems.</p><br />
<br />
<p>We settled on an <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">inducible ribo-killswitch system</a>. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example, where a deletion in the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our systems require. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation.</p><p> Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen <i>et al.</i> (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second kill gene under a common regulatory element reduces this value by two orders of magnitude. The mutation rate of a system with two kill genes under independent controls might approach 10<sup>-12</sup> (Knudsen et al 1995). Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby raising the possibility of horizontal gene transfer to other organisms.</p><br />
<br />
<br />
<br><!--<br />
<br />
<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 interviews 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 />
<br />
<h2>Design Challenges from Industry</h2><br />
<br />
<p> As we wanted our system to eventually be implemented in our tailings pond remediation system, we had several design challenges to take into account. Our interviews with industry experts helped us make </html>'''informed design choices'''<html> so as to maximize the probability of our system actually being implemented one day.</p><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.<br />
<br />
<h2> Our Solution</h2><br />
<br />
<p>With these considerations in mind, we settled on an inducible system riobo-killswitch system. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example. Here, a deletion form the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation. Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second kill gene under a common regulatory element reduces this value by two orders of magnitude. The mutation rate of a system with two kill genes under independent control elements could be as low as 10<sup>-12</sup> (Knudsen et al 1995). Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. </p><br />
<br />
<p>As industry was more interested in the transfer of genetic material over the death of the organism and we already have structural safety measures, we feel this is an appropriate solution as it responds to industry concerns. As such, our final system is a collection of novel inducible ribo-killswitchs making use of unique regulatory elements and novel exo/endonucleaes.</p><br />
<br />
<br />
<br />
<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the toxins are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer were to somehow survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if <i>E. coli</i>, 37 &deg;C; if <i>Pseudomonas</i>, 30&deg;C). In addition, we will have an agitator (turbine) and an air sparger supplying filtered air to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
<br />
<h2>Biosensor Design Considerations</h2>--><br />
</html><br />
<br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/DesignTeam:Calgary/Project/HumanPractices/Design2012-10-26T23:34:54Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Preliminary Design Considerations|<br />
CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 250px;"></img><br />
<br />
<p>FRED and OSCAR have been tasked with jobs that require them to be outside of a laboratory environment. Our discussions with industry experts emphasized the need to design a system that minimized the chance of bacteria escaping into the environment. Despite our belief that due to the increased metabolic load FRED and OSCAR are undertaking they would not be able to outcompete any native bacteria, we took these concerns to heart when we designed our project. We have designed multiple layers of controls for each system, utilizing both biological and physical controls.</p><br />
<br />
<h2>Physical: The first line of defense</h2><br />
<br />
<p>The best way to prevent FRED and OSCAR from spreading into the environment is to make sure that they cannot get to it. As such both our bioreactor and biosensor prototypes involve isolating the bacteria in closed systems. In our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">biosensor</a> we seal the tubes with a one way valve with FRED trapped inside. The tailings sample is added through the one way valve and then when the testing is done the cap is twisted slightly to release bleach into the sealed system. After the bleach is added the tube is disposed of in a safe manner.</p><br />
<br />
<p>OSCAR presents more of a challenge, as he needs to remain in one place for an extended period of time to perform his tasks. For this we have created a <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> house for him. In this sealed system filtered air is bubbled in to keep oxygen levels optimal while a HEPA filter is used to screen air coming out. To extract any hydrocarbons from the reactor a belt skimmer is used that selectively picks up oil while leaving bacteria behind. When the oil is separated from the belt skimmer it is exposed to UV to kill any bacteria, and then is placed into a fractional distillator that heats to 400&deg;C to separate the hydrocarbons from each other.</p><br />
<br />
<h2>Biological: Preparing for the worst</h2><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor to favor adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been criticized in previous iGEM systems.</p><br />
<br />
<p>We settled on an <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">inducible ribo-killswitch system</a>. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example, where a deletion in the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our systems require. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation.</p><p> Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen <i>et al.</i> (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second kill gene under a common regulatory element reduces this value by two orders of magnitude. The mutation rate of a system with two kill genes under independent controls might approach 10<sup>-12</sup> (Knudsen et al 1995). Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms.</p><br />
<br />
<br />
<br><!--<br />
<br />
<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 interviews 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 />
<br />
<h2>Design Challenges from Industry</h2><br />
<br />
<p> As we wanted our system to eventually be implemented in our tailings pond remediation system, we had several design challenges to take into account. Our interviews with industry experts helped us make </html>'''informed design choices'''<html> so as to maximize the probability of our system actually being implemented one day.</p><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.<br />
<br />
<h2> Our Solution</h2><br />
<br />
<p>With these considerations in mind, we settled on an inducible system riobo-killswitch system. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example. Here, a deletion form the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation. Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second kill gene under a common regulatory element reduces this value by two orders of magnitude. The mutation rate of a system with two kill genes under independent control elements could be as low as 10<sup>-12</sup> (Knudsen et al 1995). Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. </p><br />
<br />
<p>As industry was more interested in the transfer of genetic material over the death of the organism and we already have structural safety measures, we feel this is an appropriate solution as it responds to industry concerns. As such, our final system is a collection of novel inducible ribo-killswitchs making use of unique regulatory elements and novel exo/endonucleaes.</p><br />
<br />
<br />
<br />
<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the toxins are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer were to somehow survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if <i>E. coli</i>, 37 &deg;C; if <i>Pseudomonas</i>, 30&deg;C). In addition, we will have an agitator (turbine) and an air sparger supplying filtered air to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
<br />
<h2>Biosensor Design Considerations</h2>--><br />
</html><br />
<br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/DesignTeam:Calgary/Project/HumanPractices/Design2012-10-26T23:32:45Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Preliminary Design Considerations|<br />
CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 250px;"></img><br />
<br />
<p>FRED and OSCAR have been tasked with jobs that require them to be outside of a laboratory environment. Our discussions with industry experts emphasized the need to design a system that minimized the chance of bacteria escaping into the environment. Despite our belief that due to the increased metabolic load FRED and OSCAR are undertaking they would not be able to outcompete any native bacteria, we took these concerns to heart when we designed our project. We have designed multiple layers of controls for each system, utilizing both biological and physical controls.</p><br />
<br />
<h2>Physical: The first line of defense</h2><br />
<br />
<p>The best way to prevent FRED and OSCAR from spreading into the environment is to make sure that they cannot get to it. As such both our bioreactor and biosensor prototypes involve isolating the bacteria in closed systems. In our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">biosensor</a> we seal the tubes with a one way valve with FRED trapped inside. The tailings sample is added through the one way valve and then when the testing is done the cap is twisted slightly to release bleach into the sealed system. After the bleach is added the tube is disposed of in a safe manner.</p><br />
<br />
<p>OSCAR presents more of a challenge, as he needs to remain in one place for an extended period of time to perform his tasks. For this we have created a <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> house for him. In this sealed system filtered air is bubbled in to keep oxygen levels optimal while a HEPA filter is used to screen air coming out. To extract any hydrocarbons from the reactor a belt skimmer is used that selectively picks up oil while leaving bacteria behind. When the oil is separated from the belt skimmer it is exposed to UV to kill any bacteria, and then is placed into a fractional distillator that heats to 400&deg;C to separate the hydrocarbons from each other.</p><br />
<br />
<h2>Biological: Preparing for the worst</h2><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor to favor adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been criticized in previous iGEM systems.</p><br />
<br />
<p>We settled on an <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">inducible ribo-killswitch system</a>. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example, where a deletion in the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our systems require. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation.</p><p> Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen <i>et al.</i> (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms.</p><br />
<br />
<br />
<br><!--<br />
<br />
<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 interviews 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 />
<br />
<h2>Design Challenges from Industry</h2><br />
<br />
<p> As we wanted our system to eventually be implemented in our tailings pond remediation system, we had several design challenges to take into account. Our interviews with industry experts helped us make </html>'''informed design choices'''<html> so as to maximize the probability of our system actually being implemented one day.</p><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.<br />
<br />
<h2> Our Solution</h2><br />
<br />
<p>With these considerations in mind, we settled on an inducible system riobo-killswitch system. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example. Here, a deletion form the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation. Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second kill gene under a common regulatory element reduces this value by two orders of magnitude. The mutation rate of a system with two kill genes under independent control elements could be as low as 10<sup>-12</sup> (Knudsen et al 1995). Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. </p><br />
<br />
<p>As industry was more interested in the transfer of genetic material over the death of the organism and we already have structural safety measures, we feel this is an appropriate solution as it responds to industry concerns. As such, our final system is a collection of novel inducible ribo-killswitchs making use of unique regulatory elements and novel exo/endonucleaes.</p><br />
<br />
<br />
<br />
<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the toxins are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer were to somehow survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if <i>E. coli</i>, 37 &deg;C; if <i>Pseudomonas</i>, 30&deg;C). In addition, we will have an agitator (turbine) and an air sparger supplying filtered air to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
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<h2>Biosensor Design Considerations</h2>--><br />
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<h2>University of Calgary Support</h2><br />
<p>The University of Calgary iGEM team would like to thank and acknowledge all of the support from the University in terms of student stipends, support personnel, facilities, and materials which made this project possible. The <b>Department of Biological Sciences </b> and the <b>O'Brien Centre for the Bachelor of Health Sciences </b> provided the laboratories we worked in this summer, and the O'Brien Centre allowed us to continue our work even into the fall term. Additionally, a special thanks to all departments and faculties which financially contributed to our project.</p><br />
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<h2>Advisor and Instructor Support</h2><br />
<p>All research work was performed by the undergraduate students on our team. Our professors and advisors contributed support and ideas to the students and facilitated a safe and efficient laboratory environment. Special thanks to <b>Dr. Lisa Gieg</b>, whose experience in petroleum microbiology provided the facilities and protocols required to make our work possible (including GC/MS and culturing of various organisms). And thanks to <b>Dr. Anders Nygren</b> for his help with engineering the electrical circuits used in the our biosensor prototypes. Finally, thanks to <b>Dr. Mayi Arcellana-Panlilio</b> for her troubleshooting advice.</p><br />
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<h2>Support From Additional Professors and University Staff</h2><br />
<p>Special thanks to <b>Dr. Doug Muench</b>, one of our advisors whose support made it possible for us to obtain our laboratory space in Biological Sciences. In addition, we would like to acknowledge other professors in the department of Biological Sciences including <b>Dr. Greg Moorhead</b>, <b>Dr. C. C. Chinnappa</b> (both for supplying equipment), <b>Dr. Michael Hynes</b>, <b>Dr. Ray Turner</b>, <b>Dr. Gerrit Voordouw</b>, <b>Dr. Howard Ceri</b>, <b>Dr. Denice Bay</b>, and <b>Monika Schwering</b> for their support with supplies and protocols. Additionally we would like to recognize <b>Dr. Viola Birss</b>, <b>Bri Campbell</b>, and <b>Anusha Abhayawardhana</b> in the Department of Chemistry for their assistance with the electrochemical studies conducted by our team. Finally we would like to thank <b>Dr. Arin Sen</b> in the Department of Chemical and Petroleum Engineering for his advice with the design of our bioreactor and <b>Dr. Jennifer Cobb</b>, <b>Deirdre Lobb</b>, <b>Dr. Steve Robbins</b>, and the <b>SACRI Research Group</b> for their donation of chemicals. </p> <br />
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<h2><i>Outside of University</i> Research and Technical Support</h2><br />
<p>We would like to thank and acknowledge the support of various individuals from other Universities. This includes<br />
<b>Dr. Michael Ellison</b> for his lab's contribution of Keio Collection Knockout strains used in this project, the <b>2011 University of Washington iGEM team</b> for sending us plasmid DNA of the Petrobrick as the DNA construct available in the registry was incorrect. Thanks to <b>Dr. Josie L. Garcia</b> from the Consejo Superior de Investigaciones, Madrid Spain for contribution of a plasmid containg the <i>hpaC</i> gene. <b>Dr. John Kilbane</b> for the contribution of the <i>Rhodococcus baikonurensis</i> containing the <i>Rhodococcus</i> IGTS8 <i>dszABC</i> plasmid. Special thanks to our professor <b>Dr. Lisa Gieg</b> for her contribution of a <i>Pseudomonas sp.</i> LD2 species previously reported to degrade carbazole. Finally, special thanks to <b>Abanacai Corporation</b>, Ohio for their samples of a oil skimming sample kit used in our bioreactor design. </p><br />
<br />
<h2><i>Outside of University</i> Additional Support</h2><br />
<p>We would like to thank the individuals who we had requested to do interviews with <b>Christine Daly</b> (Suncor Inc.) and <b>Ryan Radke</b> (BioAlberta). We would like to thank our representative <b>Claudia Bustos</b> for all of her hard work and support of our team from the Telus Spark Science Centre. Thank you to <b>Robert Kotch</b> from the Bonniebrook Waste Treatment Plant for allowing us access to their facility to learn more about how their reactors were designed. We would also like to thank <b>Lorne and Laurie Swalm</b> for their generous support of our project.</p> <br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/AttributionsTeam:Calgary/Project/Attributions2012-10-26T22:31:26Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Attributions|<br />
CONTENT=<br />
<br />
<html><br />
<h2>University of Calgary Support</h2><br />
<p>The University of Calgary iGEM team would like to thank and acknowledge all of the support from the University in terms of student stipends, support personnel, facilities, and materials which made this project possible. The <b>Department of Biological Sciences </b> and the <b>O'Brien Centre for the Bachelor of Health Sciences </b> provided the laboratories we worked in this summer, and the O'Brien Centre allowed us to continue our work even into the fall term. Additionally, a special thanks to all departments and faculties which financially contributed to our project.</p><br />
<br />
<h2>Advisor and Instructor Support</h2><br />
<p>All research work was performed by the undergraduate students part of the Calgary Team. Our professors and advisors contributed support and ideas to the students and facilitated a safe and efficient laboratory environment for the students. Special thanks to <b>Dr. Lisa Gieg</b>, whose experience in petroleum microbiology provided the facilities and protocols required to make our work possible (including GC/MS and culturing of various organisms). And thanks to <b>Dr. Anders Nygren</b> for his help with engineering the electrical circuits used in the our biosensor prototypes. Finally, thanks to <b>Dr. Mayi Arcellana-Panlilio</b> for her troubleshooting advice.</p><br />
<br />
<h2>Support From Additional Professors and University Staff</h2><br />
<p>Special thanks to <b>Dr. Doug Muench</b>, one of our advisors whose support made it possible for us to obtain our laboratory space in Biological Sciences. In addition, we would like to acknowledge other professors in the department of Biological Sciences including <b>Dr. Greg Moorhead</b>, <b>Dr. C. C. Chinnappa</b> (both for supplying equipment), <b>Dr. Michael Hynes</b>, <b>Dr. Ray Turner</b>, <b>Dr. Gerrit Voordouw</b>, <b>Dr. Howard Ceri</b>, <b>Dr. Denice Bay</b>, and <b>Monika Schwering</b> for their support with supplies and protocols. Additionally we would like to recognize <b>Dr. Viola Birss</b>, <b>Bri Campbell</b>, and <b>Anusha Abhayawardhana</b> in the Department of Chemistry for their assistance with the electrochemical studies conducted by our team. Finally we would like to thank <b>Dr. Arin Sen</b> in the Department of Chemical and Petroleum Engineering for his advice with the design of our bioreactor and <b>Dr. Jennifer Cobb</b>, <b>Deirdre Lobb</b>, <b>Dr. Steve Robbins</b>, and the <b>SACRI Research Group</b> for their donation of chemicals. </p> <br />
<br />
<h2><i>Outside of University</i> Research and Technical Support</h2><br />
<p>We would like to thank and acknowledge the support of various individuals from other Universities. This includes<br />
<b>Dr. Michael Ellison</b> for his lab's contribution of Keio Collection Knockout strains used in this project, the <b>2011 University of Washington iGEM team</b> for sending us plasmid DNA of the Petrobrick as the DNA construct available in the registry was incorrect. Thanks to <b>Dr. Josie L. Garcia</b> from the Consejo Superior de Investigaciones, Madrid Spain for contribution of a plasmid containg the <i>hpaC</i> gene. <b>Dr. John Kilbane</b> for the contribution of the <i>Rhodococcus baikonurensis</i> containing the <i>Rhodococcus</i> IGTS8 <i>dszABC</i> plasmid. Special thanks to our professor <b>Dr. Lisa Gieg</b> for her contribution of a <i>Pseudomonas sp.</i> LD2 species previously reported to degrade carbazole. Finally, special thanks to <b>Abanacai Corporation</b>, Ohio for their samples of a oil skimming sample kit used in our bioreactor design. </p><br />
<br />
<h2><i>Outside of University</i> Additional Support</h2><br />
<p>We would like to thank the individuals who we had requested to do interviews with <b>Christine Daly</b> (Suncor Inc.) and <b>Ryan Radke</b> (BioAlberta). We would like to thank our representative <b>Claudia Bustos</b> for all of her hard work and support of our team from the Telus Spark Science Centre. Thank you to <b>Robert Kotch</b> from the Bonniebrook Waste Treatment Plant for allowing us access to their facility to learn more about how their reactors were designed. We would also like to thank <b>Lorne and Laurie Swalm</b> for their generous support of our project.</p> <br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/AttributionsTeam:Calgary/Project/Attributions2012-10-26T22:29:24Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Attributions|<br />
CONTENT=<br />
<br />
<html><br />
<h2>University of Calgary Support</h2><br />
<p>The University of Calgary iGEM team would like to thank and acknowledge all of the support from the University in terms of student stipends, support personnel, facilities, and materials which made this project possible. This included the <b>Department of Biological Sciences </b> and the <b>O'Brien Centre for the Bachelor of Health Sciences </b> for providing the laboratories we worked in this summer, and the O'Brien Centre for access to the laboratories even into the fall term. Additionally, a special thanks to all departments and faculties which financially contributed to our project.</p><br />
<br />
<h2>Advisor and Instructor Support</h2><br />
<p>All research work was performed by the undergraduate students part of the Calgary Team. Our professors and advisors contributed support and ideas to the students and facilitated a safe and efficient laboratory environment for the students. Special thanks to <b>Dr. Lisa Gieg</b>, whose experience in petroleum microbiology provided the facilities and protocols required to make our work possible (including GC/MS and culturing of various organisms). And thanks to <b>Dr. Anders Nygren</b> for his help with engineering the electrical circuits used in the our biosensor prototypes. Finally, thanks to <b>Dr. Mayi Arcellana-Panlilio</b> for her troubleshooting advice.</p><br />
<br />
<h2>Support From Additional Professors and University Staff</h2><br />
<p>Special thanks to <b>Dr. Doug Muench</b>, one of our advisors whose support made it possible for us to obtain our laboratory space in Biological Sciences. In addition, we would like to acknowledge other professors in the department of Biological Sciences including <b>Dr. Greg Moorhead</b>, <b>Dr. C. C. Chinnappa</b> (both for supplying equipment), <b>Dr. Michael Hynes</b>, <b>Dr. Ray Turner</b>, <b>Dr. Gerrit Voordouw</b>, <b>Dr. Howard Ceri</b>, <b>Dr. Denice Bay</b>, and <b>Monika Schwering</b> for their support with supplies and protocols. Additionally we would like to recognize <b>Dr. Viola Birss</b>, <b>Bri Campbell</b>, and <b>Anusha Abhayawardhana</b> in the Department of Chemistry for their assistance with the electrochemical studies conducted by our team. Finally we would like to thank <b>Dr. Arin Sen</b> in the Department of Chemical and Petroleum Engineering for his advice with the design of our bioreactor and <b>Dr. Jennifer Cobb</b>, <b>Deirdre Lobb</b>, <b>Dr. Steve Robbins</b>, and the <b>SACRI Research Group</b> for their donation of chemicals. </p> <br />
<br />
<h2><i>Outside of University</i> Research and Technical Support</h2><br />
<p>We would like to thank and acknowledge the support of various individuals from other Universities. This includes<br />
<b>Dr. Michael Ellison</b> for his lab's contribution of Keio Collection Knockout strains used in this project, the <b>2011 University of Washington iGEM team</b> for sending us plasmid DNA of the Petrobrick as the DNA construct available in the registry was incorrect. Thanks to <b>Dr. Josie L. Garcia</b> from the Consejo Superior de Investigaciones, Madrid Spain for contribution of a plasmid containg the <i>hpaC</i> gene. <b>Dr. John Kilbane</b> for the contribution of the <i>Rhodococcus baikonurensis</i> containing the <i>Rhodococcus</i> IGTS8 <i>dszABC</i> plasmid. Special thanks to our professor <b>Dr. Lisa Gieg</b> for her contribution of a <i>Pseudomonas sp.</i> LD2 species previously reported to degrade carbazole. Finally, special thanks to <b>Abanacai Corporation</b>, Ohio for their samples of a oil skimming sample kit used in our bioreactor design. </p><br />
<br />
<h2><i>Outside of University</i> Additional Support</h2><br />
<p>We would like to thank the individuals who we had requested to do interviews with <b>Christine Daly</b> (Suncor Inc.) and <b>Ryan Radke</b> (BioAlberta). We would like to thank our representative <b>Claudia Bustos</b> for all of her hard work and support of our team from the Telus Spark Science Centre. Thank you to <b>Robert Kotch</b> from the Bonniebrook Waste Treatment Plant for allowing us access to their facility to learn more about how their reactors were designed. We would also like to thank <b>Lorne and Laurie Swalm</b> for their generous support of our project.</p> <br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/AttributionsTeam:Calgary/Project/Attributions2012-10-26T22:28:53Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Attributions|<br />
CONTENT=<br />
<br />
<html><br />
<h2>University of Calgary Support</h2><br />
<p>The University of Calgary iGEM team would like to thank and acknowledge all of the support from the University in terms of student stipends, support personnel, facilities, and materials which made this project possible. This included the <b>Department of Biological Sciences </b> and the <b>O'Brien Centre </b> for providing the laboratories we worked in this summer, and the O'Brien Centre for access to the laboratories even into the fall term. Additionally, a special thanks to all departments and faculties which financially contributed to our project.</p><br />
<br />
<h2>Advisor and Instructor Support</h2><br />
<p>All research work was performed by the undergraduate students part of the Calgary Team. Our professors and advisors contributed support and ideas to the students and facilitated a safe and efficient laboratory environment for the students. Special thanks to <b>Dr. Lisa Gieg</b>, whose experience in petroleum microbiology provided the facilities and protocols required to make our work possible (including GC/MS and culturing of various organisms). And thanks to <b>Dr. Anders Nygren</b> for his help with engineering the electrical circuits used in the our biosensor prototypes. Finally, thanks to <b>Dr. Mayi Arcellana-Panlilio</b> for her troubleshooting advice.</p><br />
<br />
<h2>Support From Additional Professors and University Staff</h2><br />
<p>Special thanks to <b>Dr. Doug Muench</b>, one of our advisors whose support made it possible for us to obtain our laboratory space in Biological Sciences. In addition, we would like to acknowledge other professors in the department of Biological Sciences including <b>Dr. Greg Moorhead</b>, <b>Dr. C. C. Chinnappa</b> (both for supplying equipment), <b>Dr. Michael Hynes</b>, <b>Dr. Ray Turner</b>, <b>Dr. Gerrit Voordouw</b>, <b>Dr. Howard Ceri</b>, <b>Dr. Denice Bay</b>, and <b>Monika Schwering</b> for their support with supplies and protocols. Additionally we would like to recognize <b>Dr. Viola Birss</b>, <b>Bri Campbell</b>, and <b>Anusha Abhayawardhana</b> in the Department of Chemistry for their assistance with the electrochemical studies conducted by our team. Finally we would like to thank <b>Dr. Arin Sen</b> in the Department of Chemical and Petroleum Engineering for his advice with the design of our bioreactor and <b>Dr. Jennifer Cobb</b>, <b>Deirdre Lobb</b>, <b>Dr. Steve Robbins</b>, and the <b>SACRI Research Group</b> for their donation of chemicals. </p> <br />
<br />
<h2><i>Outside of University</i> Research and Technical Support</h2><br />
<p>We would like to thank and acknowledge the support of various individuals from other Universities. This includes<br />
<b>Dr. Michael Ellison</b> for his lab's contribution of Keio Collection Knockout strains used in this project, the <b>2011 University of Washington iGEM team</b> for sending us plasmid DNA of the Petrobrick as the DNA construct available in the registry was incorrect. Thanks to <b>Dr. Josie L. Garcia</b> from the Consejo Superior de Investigaciones, Madrid Spain for contribution of a plasmid containg the <i>hpaC</i> gene. <b>Dr. John Kilbane</b> for the contribution of the <i>Rhodococcus baikonurensis</i> containing the <i>Rhodococcus</i> IGTS8 <i>dszABC</i> plasmid. Special thanks to our professor <b>Dr. Lisa Gieg</b> for her contribution of a <i>Pseudomonas sp.</i> LD2 species previously reported to degrade carbazole. Finally, special thanks to <b>Abanacai Corporation</b>, Ohio for their samples of a oil skimming sample kit used in our bioreactor design. </p><br />
<br />
<h2><i>Outside of University</i> Additional Support</h2><br />
<p>We would like to thank the individuals who we had requested to do interviews with <b>Christine Daly</b> (Suncor Inc.) and <b>Ryan Radke</b> (BioAlberta). We would like to thank our representative <b>Claudia Bustos</b> for all of her hard work and support of our team from the Telus Spark Science Centre. Thank you to <b>Robert Kotch</b> from the Bonniebrook Waste Treatment Plant for allowing us access to their facility to learn more about how their reactors were designed. We would also like to thank <b>Lorne and Laurie Swalm</b> for their generous support of our project.</p> <br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/AttributionsTeam:Calgary/Project/Attributions2012-10-26T22:27:28Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Attributions|<br />
CONTENT=<br />
<br />
<html><br />
<h2>University of Calgary Support</h2><br />
<p>The University of Calgary iGEM team would like to thank and acknowledge all of the support from the University in terms of student stipends, support personnel, facilities, and materials which made this project possible. This included the Department of Biological Sciences and the O'Brien Centre for providing the laboratories we worked in this summer, and the O'Brien Centre for access to the laboratories even into the fall term. Additionally, a special thanks to all departments and faculties which financially contributed to our project.</p><br />
<br />
<h2>Advisor and Instructor Support</h2><br />
<p>All research work was performed by the undergraduate students part of the Calgary Team. Our professors and advisors contributed support and ideas to the students and facilitated a safe and efficient laboratory environment for the students. Special thanks to <b>Dr. Lisa Gieg</b>, whose experience in petroleum microbiology provided the facilities and protocols required to make our work possible (including GC/MS and culturing of various organisms). And thanks to <b>Dr. Anders Nygren</b> for his help with engineering the electrical circuits used in the our biosensor prototypes. Finally, thanks to <b>Dr. Mayi Arcellana-Panlilio</b> for her troubleshooting advice.</p><br />
<br />
<h2>Support From Additional Professors and University Staff</h2><br />
<p>Special thanks to <b>Dr. Doug Muench</b>, one of our advisors whose support made it possible for us to obtain our laboratory space in Biological Sciences. In addition, we would like to acknowledge other professors in the department of Biological Sciences including <b>Dr. Greg Moorhead</b>, <b>Dr. C. C. Chinnappa</b> (both for supplying equipment), <b>Dr. Michael Hynes</b>, <b>Dr. Ray Turner</b>, <b>Dr. Gerrit Voordouw</b>, <b>Dr. Howard Ceri</b>, <b>Dr. Denice Bay</b>, and <b>Monika Schwering</b> for their support with supplies and protocols. Additionally we would like to recognize <b>Dr. Viola Birss</b>, <b>Bri Campbell</b>, and <b>Anusha Abhayawardhana</b> in the Department of Chemistry for their assistance with the electrochemical studies conducted by our team. Finally we would like to thank <b>Dr. Arin Sen</b> in the Department of Chemical and Petroleum Engineering for his advice with the design of our bioreactor and <b>Dr. Jennifer Cobb</b>, <b>Deirdre Lobb</b>, <b>Dr. Steve Robbins</b>, and the <b>SACRI Research Group</b> for their donation of chemicals. </p> <br />
<br />
<h2><i>Outside of University</i> Research and Technical Support</h2><br />
<p>We would like to thank and acknowledge the support of various individuals from other Universities. This includes<br />
<b>Dr. Michael Ellison</b> for his lab's contribution of Keio Collection Knockout strains used in this project, the <b>2011 University of Washington iGEM team</b> for sending us plasmid DNA of the Petrobrick as the DNA construct available in the registry was incorrect. Thanks to <b>Dr. Josie L. Garcia</b> from the Consejo Superior de Investigaciones, Madrid Spain for contribution of a plasmid containg the <i>hpaC</i> gene. <b>Dr. John Kilbane</b> for the contribution of the <i>Rhodococcus baikonurensis</i> containing the <i>Rhodococcus</i> IGTS8 <i>dszABC</i> plasmid. Special thanks to our professor <b>Dr. Lisa Gieg</b> for her contribution of a <i>Pseudomonas sp.</i> LD2 species previously reported to degrade carbazole. Finally, special thanks to <b>Abanacai Corporation</b>, Ohio for their samples of a oil skimming sample kit used in our bioreactor design. </p><br />
<br />
<h2><i>Outside of University</i> Additional Support</h2><br />
<p>We would like to thank the individuals who we had requested to do interviews with <b>Christine Daly</b> (Suncor Inc.) and <b>Ryan Radke</b> (BioAlberta). We would like to thank our representative <b>Claudia Bustos</b> for all of her hard work and support of our team from the Telus Spark Science Centre. Thank you to <b>Robert Kotch</b> from the Bonniebrook Waste Treatment Plant for allowing us access to their facility to learn more about how their reactors were designed. We would also like to thank <b>Lorne and Laurie Swalm</b> for their generous support of our project.</p> <br />
<br />
</html><br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-26T22:22:02Z<p>Myarcell: </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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SIDELIST =<br />
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<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<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 />
<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’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 />
<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>Myarcellhttp://2012.igem.org/Team:Calgary/Team/AbouttheUniversityTeam:Calgary/Team/AbouttheUniversity2012-10-17T17:49:12Z<p>Myarcell: </p>
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<h2> Calgary </h2><br />
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<p>Calgary is the largest city in Alberta, and has a population of over 1 million. It was founded in 1875, when the RCMP erected Fort Calgary (then Fort Brisebois) to protect the western plains from whiskey traders. When the Canadian Pacific Railway reached the area in 1883, Calgary grew into an important commercial and agricultural center. Currently, Calgary's economy is dominated by the oil and gas industry, and major attractions include Canada Olympic Park (the site of the 1988 Winter Olympics), Calaway Park, the Lilac Festival, and the Calgary Stampede (touted as the Greatest Outdoor Show on Earth founded in 1912). Calgary is also situated near the Rocky Mountains, and plays host to a unique weather phenomenom called the chinook. Famous Calgarians include the young, incumbent mayor, Naheed Nenshi, who is the first Muslim to become mayor of a major Canadian city, as well as the current Prime Minister of Canada, Conservative leader Stephen Harper.</p><br />
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<h2>The University of Calgary</h2><br />
<p>The University of Calgary is ranked as one of Canada’s top comprehensive research institutions. Currently, more than 30,500 students are enrolled in 14 faculties in undergraduate, graduate and professional degree programs. The University contains more than 30 research institutions and centres, including the O’Brien Centre within which iGEM Calgary operates. The university is also the birthplace of many inventions including the neurochip, and has had 140,000 alumni since its founding in 1966. </p><br />
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<h2>iGEM Lab Space</h2><br />
<p>The University of Calgary iGEM team would like to thank the O' Brien Centre for the Bachelor of Health Sciences Program and the Department of Biological Sciences for providing lab space for our team. The O' Brien Centre is located in the Foothills campus of the University of Calgary. It has provided the iGEM team with lab space as well as funding ever since the University started fielding iGEM teams. The O’Brien Centre is the hub of top-notch interdisciplinary research and education. It funds approximately 9 million dollars a year for research. The department of Biological Sciences donated a teaching laboratory as lab space for our iGEM students to work in during the months of June - August. We would like to thank the entire department for their support of our team.</p><br />
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}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-04T04:05:38Z<p>Myarcell: </p>
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<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, many toxic compounds are produced. These have become a huge problem in our society resulting in 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 />
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</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 />
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<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 />
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<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 />
</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 />
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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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<br />
<p>FRED and OSCAR have been tasked with jobs that require them to be outside of a laboratory environment. Our discussions with industry experts emphasized the need to design a system that minimized the chance of bacteria escaping into the environment. Despite our belief that due to the increased metabolic load FRED and OSCAR are undertaking they would not be able to outcompete any native bacteria, we took these concerns to heart when we designed our project. We have designed multiple layers of controls for each system, utilizing both biological and physical controls.</p><br />
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<h2>Physical: The first line of defense</h2><br />
<br />
<p>The best way to prevent FRED and OSCAR from spreading into the environment is to make sure that they cannot get to it. As such both our bioreactor and biosensor prototypes involve isolating the bacteria in closed systems. In our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">biosensor</a> we seal the tubes with a one way valve with FRED trapped inside. The tailings sample is added through the one way valve and then when the testing is done the cap is twisted slightly to release bleach into the sealed system. After the bleach is added the tube is disposed of in a safe manner.</p><br />
<br />
<p>OSCAR presents more of a challenge, as he needs to remain in one place for an extended period of time to perform his tasks. For this we have created a <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> house for him. In this sealed system filtered air is bubbled in to keep oxygen levels optimal while a HEPA filter is used to screen air coming out. To extract any hydrocarbons from the reactor a belt skimmer is used that selectively picks up oil while leaving bacteria behind. When the oil is separated from the belt skimmer it is exposed to UV to kill any bacteria, and then is placed into a fractional distillator that heats to 400&deg;C to separate the hydrocarbons from each other.</p><br />
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<h2>Biological: Preparing for the worst</h2><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor to favor adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been criticized in previous iGEM systems.</p><br />
<br />
<p>We settled on an <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">inducible ribo-killswitch system</a>. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example, where a deletion in the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our systems require. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation.</p><p> Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen <i>et al.</i> (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms.</p><br />
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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 interviews 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>Design Challenges from Industry</h2><br />
<br />
<p> As we wanted our system to eventually be implemented in our tailings pond remediation system, we had several design challenges to take into account. Our interviews with industry experts helped us make </html>'''informed design choices'''<html> so as to maximize the probability of our system actually being implemented one day.</p><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.<br />
<br />
<h2> Our Solution</h2><br />
<br />
<p>With these considerations in mind, we settled on an inducible system riobo-killswitch system. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example. Here, a deletion form the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation. Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. </p><br />
<br />
<p>As industry was more interested in the transfer of genetic material over the death of the organism and we already have structural safety measures, we feel this is an appropriate solution as it responds to industry concerns. As such, our final system is a collection of novel inducible ribo-killswitchs making use of unique regulatory elements and novel exo/endonucleaes.</p><br />
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<br />
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<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the toxins are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer were to somehow survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if <i>E. coli</i>, 37 &deg;C; if <i>Pseudomonas</i>, 30&deg;C). In addition, we will have an agitator (turbine) and an air sparger supplying filtered air to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
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<h2>Biosensor Design Considerations</h2>--><br />
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<p>FRED and OSCAR have been tasked with jobs that require them to be outside of a laboratory environment. Our discussions with industry experts emphasized the need to design a system that minimized the chance of bacteria escaping into the environment. Despite our belief that due to the increased metabolic load FRED and OSCAR are undertaking they would not be able to outcompete any native bacteria, we took these concerns to heart when we designed our project. We have designed multiple layers of controls for each system, utilizing both biological and physical controls.</p><br />
<br />
<h2>Physical: The first line of defense</h2><br />
<br />
<p>The best way to prevent FRED and OSCAR from spreading into the environment is to make sure that they cannot get to it. As such both our bioreactor and biosensor prototypes involve isolating the bacteria in closed systems. In our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">biosensor</a> we seal the tubes with a one way valve with FRED trapped inside. The tailings sample is added through the one way valve and then when the testing is done the cap is twisted slightly to release bleach into the sealed system. After the bleach is added the tube is disposed of in a safe manner.</p><br />
<br />
<p>OSCAR presents more of a challenge, as he needs to remain in one place for an extended period of time to perform his tasks. For this we have created a <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> house for him. In this sealed system filtered air is bubbled in to keep oxygen levels optimal while a HEPA filter is used to screen air coming out. To extract any hydrocarbons from the reactor a belt skimmer is used that selectively picks up oil while leaving bacteria behind. When the oil is seperated from the belt skimmer it is exposed to UV to kill any bacteria, and then is placed into a fractional distillator that heats to 400&deg;C to seperate the hydrocarbons from each other.</p><br />
<br />
<h2>Biological: Preparing for the worst</h2><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.</p><br />
<br />
<p>We settled on an <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">inducible riobo-killswitch system</a>. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example, where a deletion in the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our systems require. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation.</p><p> Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen <i>et al.</i> (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms.</p><br />
<br />
<br />
<br><!--<br />
<br />
<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 interviews 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 riobo-killswitch parts. These contain riboswitch regulatory elements and exo/endonuclease kill genes.</p><br />
<br />
<h2>Design Challenges from Industry</h2><br />
<br />
<p> As we wanted our system to eventually be implemented in our tailings pond remediation system, we had several design challenges to take into account. Our interviews with industry experts helped us make </html>'''informed design choices'''<html> so as to maximize the probability of our system actually being implemented one day.</p><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.<br />
<br />
<h2> Our Solution</h2><br />
<br />
<p>With these considerations in mind, we settled on an inducible system riobo-killswitch system. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example. Here, a deletion form the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation. Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. </p><br />
<br />
<p>As industry was more interested in the transfer of genetic material over the death of the organism and we already have structural safety measures, we feel this is an appropriate solution as it responds to industry concerns. As such, our final system is a collection of novel inducible ribo-killswitchs making use of unique regulatory elements and novel exo/endonucleaes.</p><br />
<br />
<br />
<br />
<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the toxins are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer were to somehow survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if <i>E. coli</i>, 37 &deg;C; if <i>Pseudomonas</i>, 30&deg;C). In addition, we will have an agitator (turbine) and an air sparger supplying filtered air to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
<br />
<h2>Biosensor Design Considerations</h2>--><br />
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}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/DesignTeam:Calgary/Project/HumanPractices/Design2012-10-04T03:11:57Z<p>Myarcell: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Preliminary Design Considerations|<br />
CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 250px;"></img><br />
<br />
<p>FRED and OSCAR have been tasked with jobs that require them to be outside of a laboratory environment. Our discussions with industry experts emphasized the need to design a system that minimized the chance of bacteria escaping into the environment. Despite our belief that due to the increased metabolic load FRED and OSCAR are undertaking they would not be able to outcompete any native bacteria, we took these concerns to heart when we designed our project. We have designed multiple layers of controls for each system, utilizing both biological and physical controls.</p><br />
<br />
<h2>Physical: The first line of defense</h2><br />
<br />
<p>The best way to prevent FRED and OSCAR from spreading into the environment is to make sure that they can't get to it. As such both our bioreactor and biosensor prototypes involve isolating the bacteria in closed systems. In our <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">biosensor</a> we seal the tubes with a one way valve with FRED trapped inside. The tailings sample is added through the one way valve and then when the testing is done the cap is twisted slightly to release bleach into the sealed system. After the bleach is added the tube is disposed of in a safe manner.</p><br />
<br />
<p>OSCAR presents more of a challenge, as he needs to remain in one place for an extended period of time to perform his tasks. For this we have created a <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> house for him. In this sealed system filtered air is bubbled in to keep oxygen levels optimal while a HEPA filter is used to screen air coming out. To extract any hydrocarbons from the reactor a belt skimmer is used that selectively picks up oil while leaving bacteria behind. When the oil is seperated from the belt skimmer it is exposed to UV to kill any bacteria, and then is placed into a fractional distillator that heats to 400&deg;C to seperate the hydrocarbons from each other.</p><br />
<br />
<h2>Biological: Preparing for the worst</h2><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.</p><br />
<br />
<p>We settled on an <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">inducible riobo-killswitch system</a>. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example, where a deletion in the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our systems require. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation.</p><p> Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen <i>et al.</i> (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms.</p><br />
<br />
<br />
<br><!--<br />
<br />
<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 interviews 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 riobo-killswitch parts. These contain riboswitch regulatory elements and exo/endonuclease kill genes.</p><br />
<br />
<h2>Design Challenges from Industry</h2><br />
<br />
<p> As we wanted our system to eventually be implemented in our tailings pond remediation system, we had several design challenges to take into account. Our interviews with industry experts helped us make </html>'''informed design choices'''<html> so as to maximize the probability of our system actually being implemented one day.</p><br />
<br />
<p>Although industry experts felt a genetic safety mechanism was important, they felt that that it needed to fit into a cost effective remediation solution. It was stressed that price is a paramount factor in permitting adoption at industrial scales, and so an inexpensive system would have a greater likelihood of being implemented outside the laboratory. Experts were also more concerned about the spread of DNA over the death of our organisms. As such, we needed a system where we avoided lysis of our cells, so as to prevent possible uptake of DNA by other surrounding organisms in soil and water, something that has been a critique of previous iGEM systems.<br />
<br />
<h2> Our Solution</h2><br />
<br />
<p>With these considerations in mind, we settled on an inducible system riobo-killswitch system. Although inducible systems have been shown to have a tendency to mutate, rendering them ineffective and allowing possible escape, they are more cost effective than other strategies. An auxotrophic marker could have been used for example. Here, a deletion form the genome would make the organism dependent on an externally supplemented metabolite. Although a mutation restoring the metabolite would be sufficiently complex as to be rendered improbable, metabolic supplements are considerably more expensive than the glucose and metal ions that our system requires. As such, we used an inducible system, but took several approaches so as to mitigate the risk of mutation. Firstly, we engineered redundancy into our system. By using two kill genes, both would have to be rendered inoperable for the kill switch mechanism to malfunction. Knudsen et al. (1995) proposed that active kill switches containing a single kill element were subject to a mutation rate of 10<sup>-6</sup> per cell per generation, but that a second redundant kill gene reduces this value by two orders of magnitude. Secondly, the kill switch mechanism is, of course, only a failsafe measure for controlling our organism's spread. The primary means of preventing its escape is through the multiple layers of mechanical security provided by our bioreactor and biosensor. Only when these measures fail will the kill switch be required to function. We felt that this system would also mitigate the concern that an auxotrophic control mechanism would kill the organism without degrading its genetic material, thereby making possible horizontal gene transfer to other organisms. </p><br />
<br />
<p>As industry was more interested in the transfer of genetic material over the death of the organism and we already have structural safety measures, we feel this is an appropriate solution as it responds to industry concerns. As such, our final system is a collection of novel inducible ribo-killswitchs making use of unique regulatory elements and novel exo/endonucleaes.</p><br />
<br />
<br />
<br />
<h2>Bioreactor Design Considerations</h2><br />
<p>Over the summer, much thought was put into the design of our bioreactor in order to optimize functionality, expense, and safety. Although many of the details of our design cannot be worked out due to the time constraint of a four-month period, there are still lots of theoretical aspects that we were able to cover.<br />
</p><br />
<p>The first aspect of our design was choosing what type of bioreactor system to use. For lab scale experiments and design, we chose to use a system that is closest to that of a batch system. This system requires all reactants to be added at time zero, with everything being removed at once when the remediation has come to completion. However, our design uses a belt skimmer to continually remove any products (hydrocarbons) formed either emulsified or found on the top layer. This way, we are able to reuse our culture and remove product until all the toxins are converted. We then remove everything in the tank and begin the process again. Our skimmed product goes through UV radiation in order to kill any bacteria that happen to be left in the product. <br />
<br />
In addition, our bioreactor bacteria will contain kill genes. When our bacteria are in a glucose-free environment (a.k.a. outside the tank) the bacteria are programmed to self-destruct. Since we have three different intermediate steps for remediation (desulphurization, denitrification, decarboxylation) we will need three tanks with the product from the previous tank acting as the reactant for the next tank in line. The product of the last tank will go through distillation to purify our desired alkane. Distillation will also assist in classifying the different hydrocarbons we formed and ensure bacteria do not escape into the environment. The produced hydrocarbons may have different carbon and hydrogen bonds, thus its boiling and condensation temperature will vary. If the bacteria removed by the belt skimmer were to somehow survive UV radiation and thrive in a glucose-free environment, it would be distilled along with the rest of the skimmed material. In distillation the bacteria would be heated to an extremely high temperature and would consequently die as a result.<br />
</p><br />
<p>To improve the growth and environment of our bacteria, we will keep our bioreactor at ideal growth temperature (if <i>E. coli</i>, 37 &deg;C; if <i>Pseudomonas</i>, 30&deg;C). In addition, we will have an agitator (turbine) and an air sparger supplying filtered air to help mix and oxygenate our solution. In order to help maintain these ideal conditions, our bioreactor will be a closed system with our belt skimmer contained inside the tank.</p><br />
<br />
<h2>Biosensor Design Considerations</h2>--><br />
</html><br />
<br />
}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-04T03:09:24Z<p>Myarcell: </p>
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<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<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 />
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<h2>BioAlberta's Ryan Radke on Biology in the Oil Sands</h2><br />
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<h2>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h2><br />
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}}</div>Myarcellhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-04T03:07:02Z<p>Myarcell: </p>
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<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
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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 />
</p><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 />
<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 />
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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 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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<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 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 />
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<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|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 />
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<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 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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