http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=MaggieRY2012.igem.org - User contributions [en]2024-03-28T22:32:06ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:53:33Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Post-Regionals">Post-Regionals</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
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TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
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<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
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<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
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<iframe width="640" height="360" src="http://www.youtube.com/embed/tkwafeHnG-U" frameborder="0" allowfullscreen></iframe><br />
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<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
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<br />
<p>In short, the bioreactor was filled with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be used for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing an <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:52:17Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|proj_hp|<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
<br />
<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be used for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing an <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:51:31Z<p>MaggieRY: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
<br />
<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be used for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing a <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:49:25Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
<br />
<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
<br />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<br />
</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
<br />
<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
<br />
<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
<br />
</html>[[File:Calgary Rha S7 Data.png|thumb|500px|center|Figure 2: pRHA-S7 construct demonstrating our kill switch in TOP10 wild type cells and <i>glyA</i> knockout cells. This demonstrates that our system is capable of being induced by the sugar rhamnose and repressed in the presence of glucose. There is no growth in rhamnose with our system as the <i>RhaBAD</i> operon has been deleted in the knockout strain we are using.]]<html><br />
<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. Current change over time illustrating <i>lacZ</i> induction by our identified transposon sensory element in a tailings pond water sample. The blue curve represents the tailings water test while the red curves shows the basal expression of the sensory element without tailings pond water present. This shows that our transposon clone has the ability to sense something within tailings pond water samples. ]]<html><br />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
<br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/4NcOKCwHCBI" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<br />
<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be turn on this device to allow for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
<br />
<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing a <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:48:33Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|proj_hp|<br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we had made on our project with us. We got a variety of different perspectives from suggestions on the scale up of our project, to the cost and environmental impact of our numerous components. The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, OSCAR components, and killswitch components, but how functional are these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> to put these pieces together in order to assess how far we have actually gotten</u>.</p><br />
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<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our identified transposon. Finally, with OSCAR we show that <b>our killswitch auxotroph dramatically increases the production of hydrocarbons in the system</b> and that we are capable of <b>scaling up</b> OSCAR's bioreactor and selectively collect hydrocarbons with our belt skimmer device.</p><br />
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<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
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<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
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<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
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<p>To test if using the <i>glyA</i> knockout strain in conjunction with our kill switch was effective, we transformed our Prha-S7 construct into the knockout strain as shown in Figure 2.</p><br />
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<p>This data suggests that our killswitch system can act synergistically with the glycine auxotroph. In the prescence of glucose you see growth of both TOP10 and <i>glyA</i> knockout cells showing that our system is repressed. There is less growth in our glycine knockout as there was not a significant amount of glycine used in the media. The TOP10 control cell line did not show growth over 24 hours which was likely due to error in the read. In the presence of rhamnose, the kill switch is capable of being induced in both TOP10 and glycine knockout strains as shown by the decrease in CFU counts. This demonstrates a functional kill switch mechanism with the Prha promoter and auxotroph.</p><br />
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<h2> <u>Putting FRED together</u> </h2><br />
<h2>Can we sense toxins?</h2><br />
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<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. To do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. The results of this assay can be shown below.</p><br />
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<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
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<h2> Taking FRED out to the field! </h2><br />
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<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
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<p> We also created a video to show how we would test this water sample with our prototype and software package. This video can be found below.</p><br />
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<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact it's better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.</p><br />
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</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure 4: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
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<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
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<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be turn on this device to allow for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
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</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure 5: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure 6: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
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<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing a <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
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<br><h2>Why Choose Hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
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<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of disrupting metabolism or the limitations of a single channel output.</p><br />
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<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
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[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
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<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
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<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
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<h2>Genes, Chemicals, and Circuits</h2><br />
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<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
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[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
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<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
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[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
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<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
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<h2>Does it Work?</h2><br />
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<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
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[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
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<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
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<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
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<h2>What Next?</h2><br />
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<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
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</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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</html><br />
[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
<br />
<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
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</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
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</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
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<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
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<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
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<h3> Results </h3><br />
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<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
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<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
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<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
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<h3> Kill Gene Testing </h3><br />
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<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
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<h3> Results </h3><br />
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</html>[[File:24 hour assay with mgtAp-mgtArb-S7 Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
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<h2>An alternative: a glucose repressible system</h2><br />
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<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
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</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T02:25:29Z<p>MaggieRY: </p>
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TITLE=Regulation/Expression Platform|<br />
CONTENT=<br />
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<img src="https://static.igem.org/mediawiki/2012/8/8c/UCalgary2012_FRED_Killswitch_Regulation_Low-Res.png" style="float: right; padding: 10px; height: 200px;"></img><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
<br />
</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
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<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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</html><br />
[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
<br />
</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<br />
<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
<br />
</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
<br />
<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
<br />
<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
<br />
<h3> Results </h3><br />
<br />
<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
<br />
<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
<br />
<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
<br />
<h3> Kill Gene Testing </h3><br />
<br />
<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
<br />
<h3> Results </h3><br />
<br />
</html>[[File:24 hour assay with mgtAp-mgtArb-S7 Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
<br />
<br />
<h2>An alternative: a glucose repressible system</h2><br />
<br />
<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
<html><br />
<br />
</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
<html><br />
<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T02:20:52Z<p>MaggieRY: </p>
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TITLE=Regulation/Expression Platform|<br />
CONTENT=<br />
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<img src="https://static.igem.org/mediawiki/2012/8/8c/UCalgary2012_FRED_Killswitch_Regulation_Low-Res.png" style="float: right; padding: 10px; height: 200px;"></img><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
<br />
</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
<br />
<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
<br />
<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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</html><br />
[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
<br />
<h2> The Moco Riboswitch </h2><br />
<br />
<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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<br />
</html><br />
[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
<br />
</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<br />
<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
<br />
<h3> GFP testing</h3><br />
<br />
</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
<br />
<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
<br />
<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
<br />
<h3> Results </h3><br />
<br />
<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
<br />
<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
<br />
<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
<br />
<h3> Kill Gene Testing </h3><br />
<br />
<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
<br />
<h3> Results </h3><br />
<br />
</html>[[File:24 hour assay with <i>mgtAp-mgtArb-S7</i> Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
<br />
<br />
<h2>An alternative: a glucose repressible system</h2><br />
<br />
<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T01:40:38Z<p>MaggieRY: </p>
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
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</html> [[File:UofC_RIBOSWITCH.png|centre|350px]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 1: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
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<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 2: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
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<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 3. </p><br />
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</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 3: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
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</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 4: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
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<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 4 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
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<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
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<h3> Results </h3><br />
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<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 5. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 5: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
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<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
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<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
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<h3> Kill Gene Testing </h3><br />
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<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
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<h3> Results </h3><br />
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</html>[[File:24 hour assay with mgtap-rb-S7 Ucalgary.png|thumb|750px|center| Figure 6: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 6 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
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<h2>An alternative: a glucose repressible system</h2><br />
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<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 7). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 7: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 8: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our flux variability analysis showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our Synergy Page for more information. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T01:08:43Z<p>MaggieRY: </p>
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
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</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
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<h2>Incorporating human practices in the design of our system </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’ve made on our project with us. We got a variety of different perspectives from suggestions on the...... The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, we have OSCAR components, and we have some killswitch components, but how functional are some of these parts, and how do they work together. So our next major goal was to <b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d actually gotten.</p><br />
<br />
<h2> Putting FRED together </h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given hat</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<h2> Putting together our killswitches </h2><br />
<br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. So we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2> Putting our Killswitch into OSCAR</h2><br />
<br />
<p>The next thing we wanted to validate was that our glycine knockout strain would in fact work as we wanted it to in OSCAR. Namely, we wanted to know if putting the PetroBrick into our glycine knockout strain and growing it in the presence of glycine would still give us the same increased hydrocarbon production that we saw when validating our model. We transformed the PetroBrick into the knockout strain and repeated the PetroBrick validation assay protocol. Our results are shown below:</p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to to perform a kind of field test with FRED, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be absolutely assured that it won’t be released. However, we could test tailings water with our biosensor prototype in the lab. Here is the data for this test. </p><br />
<br />
<H2> Putting OSCAR in action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached Kelly Roberge, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (1000L+ tanks). To start with, we would need to consider the amount of naphthenic acids needed to provide steady throughput in our system and just how much hydrocarbon can be produced in a full cycle of our system. He recommended that we use computer modelling to explore these challenges. This could allow us to determine the possible hydrocarbon output of our lab scale experiments once they are up and running. Additionally, we would need to take into consideration the composition of tailings pond solution, especially the sludge and bitumen content. The sludge could be physically harmful to our bioreactor and reduce its overall efficiency as well. A possible way to tackle this challenge would be to use current mature fine tailings drying techniques used to help speed the reuse of water in the tailings ponds. As tailings fines settle the resulting tailings water component would be left behind. This would be an ideal input into our system for potential remediation and production of hydrocarbons as it would contain a large proportion of the compounds thought to be most toxic in the tailings. By using this matured tailings as the input to our system it could help increase the efficiency of our bioreactor and provide for a smoother scale up from the lab bench to an industrial bioreactor.</p><br />
<br />
<h2>Glycine Auxotrophy Still Allows For Hydrocarbon Production</h2><br />
<p><b>In fact its better!</b> The glycine auxotroph will be used as a second layer of regulation with our kill switch in the event that our bacterium is capable of escaping the bioreactor. However in order to ensure that the glycine knockout we are using does not compromise the production of hydrocarbons and we can continue to see the high yield of hydrocarbons as predicted with our flux balance modelling, we performed an experiment to look at the relative amount of hydrocarbon production as in the flux balance analysis model. As seen in the figure below, using the <i>glyA</i> knockout greatly increased the output of hydrocarbons much higher than in the wild type <i>E. coli</i> strain. This was extremely exciting showing that our system could not only be safe, with a second layer of control for safety, and an increase in output.<br />
<br />
</html>[[File:Calgary glyAKOPetrobrick.png|thumb|500px|center|Figure X: Relative production of hydrocarbons per cell as discussed in the flux balance analysis section of our wiki. Wild type <i>E. coli</i> TOP10 cells were incubated with minimal media 1% glucose (Negative) or 50:50 LB:Washington Production Media (Positive). Additionally, the <i>glyA</i> knockout was incubated in minimal media in the presence of glycine. Production of C15 hydrocarbon was standardized to OD<sub>600</sub> measurements and normalized to the positive control. Surprisingly, the <i>glyA</i> knockout greatly increased the amount of hydrocarbons (almost 3x the amount of hydrocarbons per cell) produced compared to both controls.]]<html><br />
<br />
<br />
</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure X: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure X: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-26T23:49:39Z<p>MaggieRY: </p>
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<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the industry where it would be employed. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
</p><br />
<br />
<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
<p>We spoke with Christine Daly, an Aquatic Reclamation Research Coordinator at Suncor Energy Inc. Christine expressed an interest in our <a href="https://2011.igem.org/Team:Calgary">project in 2011</a> and was willing to discuss this year’s project design with us. One major point that was brought up early on in our design was that there is an opportunity for engineered organisms to outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar concern. To address these concerns, we created our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> system, which would physically contain our bacteria, and also a <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">genetic killswitch mechanism</a>. Another interesting point brought up in this discussion was how the oil industry is currently looking into biology as one of many potential alternative methods to remediate the toxic components of tailings ponds and the oil sands in general. Research exists with other systems such as algal bioremediation, but practical implementations of biology in the oil sands appear to be rather few and far between. Oil industries do, however, appear to show an increased interest in biology (and in turn, synthetic biology) as a possible solution to various problems, a sentiment reflected in <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">our dialogue with the Oil Sands Leadership Initiative</a>.</p><br />
<p>The full interview can be viewed below.</p><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/GiM6EIC9XBo" frameborder="0" allowfullscreen></iframe><br />
<br />
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<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/86XQ-Kg5fJ4" frameborder="0" allowfullscreen></iframe><br />
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<br />
<a name="postregionals"></a><br />
<h2>Follow-Up Interviews</h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
<br />
<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
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<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-26T20:43:03Z<p>MaggieRY: </p>
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
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TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<br />
<h2>Incorporating human practices in the design of our system </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. In order to ensure we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’ve made on our project with us. We got a variety of different perspectives from suggestions on the...... The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, we have OSCAR components, and we have some killswitch components, but how functional are some of these parts, and how do they work together. So our next major goal was to <b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d actually gotten.</p><br />
<br />
<h2> Putting FRED together </h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given hat</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<h2> Putting together our killswitches </h2><br />
<br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. So we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2> Putting our Killswitch into OSCAR</h2><br />
<br />
<p>The next thing we wanted to validate was that our glycine knockout strain would in fact work as we wanted it to in OSCAR. Namely, we wanted to know if putting the PetroBrick into our glycine knockout strain and growing it in the presence of glycine would still give us the same increased hydrocarbon production that we saw when validating our model. We transformed the PetroBrick into the knockout strain and repeated the PetroBrick validation assay protocol. Our results are shown below:</p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that FRED could actually detect toxins found in tailings ponds within a laboratory setting, the next challenge would be to take FRED out to a tailings pond to out him to the test. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. We still felt that testing FRED out in the real world, demonstrating that our prototype was easy to use and functional outside of a lab environment was extremely important. As such, we decided to do a field test of a body of water in our own city. The first thing we worried about though, was if there was any regulation surrounding water sampling, or performing a field test with a genetically modified organism (GMO). So we did a literature search to look for any regulations that might exist. We couldn’t find anything that pertained to our province, so we looked to Ontario and the United States. We looked at the concise guide to U.S. federal guidelines, rules and regulations for synthetic biology. In this guide, rules pertaining to field tests are covered. In cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification.</p><br />
<br />
<H2> Putting OSCAR in action! </h2><br />
<p> Once we had tested FRED and showed that he is not only able to , we wanted to put OSCAR into action and who that the design of our bioreactor was capable of doing what we wanted it to. By the end of the summer, we had a lab scale prototype and design for our bioreactor. To help us move forward with this portion of the project we interviewed Kelly Roberge, a consultant for oil sands specializing in tailings ponds. This interview gave us much to think about and helped us form ideas on how to improve the overall design of our bioreactor. In particular, Kelly’s advice and questions surrounded scaling up our bioreactor to industrial size. <br />
There are many things to consider when going from lab scale to industrial scale, and very little can be correlated linearly when moving from lab scale to industrial size (1000L+ tanks). To start, we would have to consider the amount of naphthenic acids needed to provide steady throughput in our system, and how much hydrocarbon can be produced in a full cycle of our system. To help provide theoretical solutions to these issues, we could determine the hydrocarbon output of our lab scale experiments once they are up and running to get an idea of what kind of numbers we are dealing with.<br />
In addition, we will have to take into consideration the composition of tailings pond solution, especially the sludge and bitumen content. This sludge could be harmful to our bioreactor and reduce the efficiency as well. One way we could solve this problem is by utilizing current NFT drying techniques used to help degrade tailings ponds. A sludge reduced water runoff is the result of this process. This water runoff could be the input to our system, which still contains large quantities of naphthenic acids. This would help increase the efficiency of our bioreactor and even allow scale up to be possible. </p><br />
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
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<h2>Incorporating human practices in the design of our system </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. In order to ensure we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’ve made on our project with us. We got a variety of different perspectives from suggestions on the...... The results of all of these can be found on our <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Interviews</b></a> page. One major concern was <b>scale-up</b>. One expert wanted to know how feasible this system would actually be. We have some FRED components, we have OSCAR components, and we have some killswitch components, but how functional are some of these parts, and how do they work together. So our next major goal was to <b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d actually gotten.</p><br />
<br />
<h2> Putting FRED together </h2><br />
<br />
<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given hat</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<h2> Putting together our killswitches </h2><br />
<br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. So we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
<br />
<h2> Putting our Killswitch into OSCAR</h2><br />
<br />
<p>The next thing we wanted to validate was that our glycine knockout strain would in fact work as we Wanted it to in OSCAR. Namely, we wanted to know if putting the PetroBrick into our glycine knockout strain and growing it in the presence of glycine would still give us the same increased hydrocarbon production that we saw when validating our model. We transformed the PetroBrick into the knockout strain and repeated the PetroBrick validation assay protocol. Our results are shown below:</p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that FRED could actually detect toxins found in tailings ponds within a laboratory setting, the next challenge would be to take FRED out to a tailings pond to out him to the test. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. We still felt that testing FRED out in the real world, demonstrating that our prototype was easy to use and functional outside of a lab environment was extremely important. As such, we decided to do a field test in a body of water in our own city. The first thing we worried about though, was if there was any regulation surrounding water sampling, or performing a field test with a genetically modified organism (GMO). So we did a literature search to look for any regulations that might exist. We couldn’t find anything that pertained to our province, so we looked to Ontario and the United States. We looked at the concise guide to U.S. federal guidelines, rules and regulations for synthetic biology. In this guide, rules pertaining to field tests are covered. In cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification.</p><br />
<br />
<H2> Putting OSCAR in action! </h2><br />
<p> Once we had tested FRED and showed that he is not only able to , we wanted to put OSCAR into action and who that the design of our bioreactor was capable of doing what we wanted it to. By the end of the summer, we had a lab scale prototype and design for our bioreactor. To help us move forward with this portion of the project we interviewed Kelly Roberge, a consultant for oil sands specializing in tailings ponds. This interview gave us much to think about and helped us form ideas on how to improve the overall design of our bioreactor. In particular, Kelly’s advice and questions surrounded scaling up our bioreactor to industrial size. <br />
There are many things to consider when going from lab scale to industrial scale, and very little can be correlated linearly when moving from lab scale to industrial size (1000L+ tanks). To start, we would have to consider the amount of naphthenic acids needed to provide steady throughput in our system, and how much hydrocarbon can be produced in a full cycle of our system. To help provide theoretical solutions to these issues, we could determine the hydrocarbon output of our lab scale experiments once they are up and running to get an idea of what kind of numbers we are dealing with.<br />
In addition, we will have to take into consideration the composition of tailings pond solution, especially the sludge and bitumen content. This sludge could be harmful to our bioreactor and reduce the efficiency as well. One way we could solve this problem is by utilizing current NFT drying techniques used to help degrade tailings ponds. A sludge reduced water runoff is the result of this process. This water runoff could be the input to our system, which still contains large quantities of naphthenic acids. This would help increase the efficiency of our bioreactor and even allow scale up to be possible. </p><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-26T09:53:55Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similarly sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|Figure 1: Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the bioreactors. ]]<html><br />
<p>Before diving into making a bioreactor, we first had to research current solutions in the field. To help us with this phase, we read papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to be considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors doing research on bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Here is a picture from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable was a cross between a fed-batch system and a continuous stir method in a closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. To remove the hydrocarbons from the culture we decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons, which is favored for an industrial scale (1000+ L tanks). Tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p> <br />
<br />
<p>To make sure that the belt does not transfer live bacteria into the hydrocarbon collection tank, we will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of radiation just before the hydrocarbons are removed from the bioreactor chamber.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|Figure 2: From computer to prototype: how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took shape using materials we found in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is the first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. Since our bioreactor would necessarily have live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
<br />
<h2>The Belt Skimmer in Action</h2><br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video showcase our belt skimmer. The model hydrocarbons stick to the belt shown in the video and are skimmed off into a Tim Horton's coffee cup (the only disposable cup we could find in the lab. </p><br />
<br />
<h2>Current Prototype with both the Sparger and Turbine</h2><br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> The second video shows our bioreactor prototype in action with both the sparger and turbine running. </p><br />
<br />
<h2>Belt Skimmer with a Collection Chamber</h2><br />
<div align="center"><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/HtcgPG3reH4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video shows the collection chamber we added to the bioreactor. There is a hole at the bottom of the chamber so that during the bioreactors' showcase the model hydrocarbons don't accumulate in the chamber. </p><br />
<br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using the Maya and RealFlow programs. Maya is a complex and extremely versatile computer animation program used in many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/m4Lz6Z8HiQA" frameborder="0" allowfullscreen></iframe><br />
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<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
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<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/Hm0r9xw9Zcw" frameborder="0" allowfullscreen></iframe><br />
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<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
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<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|Figure 3: Left panel: Belt rankings from three different tests. We tested the ability of the belts to pick up hydrocarbons, and to exclude bacteria or tailings pond water. Right panel: The five belts we tested and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution so as to prevent the settling of bacterial cells and other heavier materials and to ensure even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for aerobic bacteria to thrive. When assembled together, the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger, a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. In order to use the air sparger, we decided to use a HEPA filter to screen air coming out of the system to maintain constant pressure in the tank. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|Figure 4: This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was a very encouraging result since we want to skim the hydrocarbon products from the top layer of our bioreactor.]]<html><br />
<br />
<p> Furthermore, we tested our belts' ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, many different types of NA's were represented in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This data suggests that minimal NA's were found in our skimmed hydrocarbon layer and that most were left in the water layer. <br />
</html>[[File:HC layer, skimmed (no NaOH)-ucalgary.png|thumb|600px|center|Figure 5: This graph shows the amount of NA's found in the skimmed hydrocarbon layer. Each bar represents the carbon ring count for a different type of NA. Clearly, minimal NA's were skimmed into the hydrocarbon layer.]]<html><br />
</html>[[File:Water layer, skimmed-ucalgary.png|thumb|600px|centre|Figure 6: This graph shows how many NA's were left in the water layer of our skimmed solution. This data suggests that minimal amounts of NA were skimmed into our solution, and with most of those skimmed found in the water layer.]] <html><br />
<br />
<br />
<h2>The Final System</h2><br />
<p>Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR bacterium would have the most suitable kill-switch circuit attached to its respective hydrocarbon conversion circuit. The bacterium would also have a deletion of a gene for the biosynthesis of glycine. Glycine would be supplemented in our defined production media, but cells would not survive outside of the bioreactor where glycine is absent. We envision OSCAR to be a co-culture of decarboxylation, decatecholization, denitrogenation, and desulfurization. Lastly, due to the energetically expensive nature of maintaining the circuits, we anticipate that if the circuits are constitutively produced cell growth rate may be very slow. Therefore in the final circuits we may want them to be activated by quorum sensing promoter systems. Essentially, when cells are at low density they focus energy on growth; when cells reach appropriate density for the reaction chamber, transcription of the circuit is enabled. Together we hope that the system will clean up recalcitrant petroleum waste and produce energy.<br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-26T09:49:47Z<p>MaggieRY: </p>
<hr />
<div>[http://www.example.com link title]{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=Bioreactor: The House of OSCAR|<br />
<br />
CONTENT=<br />
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<html><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/UCalgary2012_OSCAR_Bioreactor_Low-Res.png" style="float:right; max-width: 200px; padding: 10px;"></img><br />
<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similarly sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
</p><br />
<br />
<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|Figure 1: Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the bioreactors. ]]<html><br />
<p>Before diving into making a bioreactor, we first had to research current solutions in the field. To help us with this phase, we read papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to be considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors doing research on bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Here is a picture from our trip to the wastewater plant!</p><br />
<br />
<br />
<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable was a cross between a fed-batch system and a continuous stir method in a closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. To remove the hydrocarbons from the culture we decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons, which is favored for an industrial scale (1000+ L tanks). Tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p> <br />
<br />
<p>To make sure that the belt does not transfer live bacteria into the hydrocarbon collection tank, we will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of radiation just before the hydrocarbons are removed from the bioreactor chamber.</p><br />
<br />
</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|Figure 2: From computer to prototype: how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took shape using materials we found in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is the first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
<br />
<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. Since our bioreactor would necessarily have live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
<br />
<h2>The Belt Skimmer in Action</h2><br />
<div align="center"><br />
<iframe width="600" height="338" align="center" src="http://www.youtube.com/embed/DVTR68DMi5U" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video showcase our belt skimmer. The model hydrocarbons stick to the belt shown in the video and are skimmed off into a Tim Horton's coffee cup (the only disposable cup we could find in the lab. </p><br />
<br />
<h2>Current Prototype with both the Sparger and Turbine</h2><br />
<div align="center"><br />
<iframe width="338" height="600" align="center" src="http://www.youtube.com/embed/4onfIfuQJ9c" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> The second video shows our bioreactor prototype in action with both the sparger and turbine running. </p><br />
<br />
<h2>Belt Skimmer with a Collection Chamber</h2><br />
<div align="center"><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/HtcgPG3reH4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<p> This video shows the collection chamber we added to the bioreactor. There is a hole at the bottom of the chamber so that during the bioreactors' showcase the model hydrocarbons don't accumulate in the chamber. </p><br />
<br />
<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using the Maya and RealFlow programs. Maya is a complex and extremely versatile computer animation program used in many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
<br />
<h2>Particle Simulation Using RealFlow2012</h2><br />
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<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
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<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
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<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|Figure 3: Left panel: Belt rankings from three different tests. We tested the ability of the belts to pick up hydrocarbons, and to exclude bacteria or tailings pond water. Right panel: The five belts we tested and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution so as to prevent the settling of bacterial cells and other heavier materials and to ensure even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for aerobic bacteria to thrive. When assembled together, the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger, a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. In order to use the air sparger, we decided to use a HEPA filter to screen air coming out of the system to maintain constant pressure in the tank. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|Figure 4: This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was a very encouraging result since we want to skim the hydrocarbon products from the top layer of our bioreactor.]]<html><br />
<br />
<p> Furthermore, we tested our belts' ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, many different types of NA's were represented in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This data suggests that minimal NA's were found in our skimmed hydrocarbon layer and that most were left in the water layer. <br />
</html>[[File:HC layer, skimmed (no NaOH)-ucalgary.png|thumb|600px|center|Figure 5: This graph shows the amount of NA's found in the skimmed hydrocarbon layer. Each bar represents the carbon ring count for a different type of NA. Clearly, minimal NA's were skimmed into the hydrocarbon layer.]]<html><br />
</html>[[File:Water layer, skimmed-ucalgary.png|thumb|600px|centre|Figure 6: This graph shows how many NA's were left in the water layer of our skimmed solution. This data suggests that minimal amounts of NA were skimmed into our solution, and with most of those skimmed found in the water layer.]] <html><br />
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<br />
<h2>The Final System</h2><br />
<p>Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR bacterium would have the most suitable kill-switch circuit attached to its respective hydrocarbon conversion circuit. The bacterium would also have a deletion of a gene for the biosynthesis of glycine, which would be supplemented in our defined production media, but would prohibit survival outside of the bioreactor. We envision OSCAR to be a co-culture of decarboxylation, decatecholization, denitrogenation, and desulfurization. Lastly, due to the energetically expensive nature of maintaining the circuits, we anticipate that if the circuits are constitutively produced cell growth rate may be very slow. Therefore in the final circuits we may want them to be activated by quorum sensing promoter systems. Essentially, when cells are at low density they focus energy on growth; when cells reach appropriate density for the reaction chamber, transcription of the circuit is enabled. Together we hope that the system will clean up recalcitrant petroleum waste and produce energy.<br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/OSCAR/BioreactorTeam:Calgary/Project/OSCAR/Bioreactor2012-10-26T09:46:50Z<p>MaggieRY: </p>
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<h2>Introduction</h2><br />
<p>We want to use the genetically engineered bacteria of the OSCAR project to convert toxic organic compounds into recoverable hydrocarbons. To accomplish this goal our team has designed a contained bioreactor system to operate in the locations of oil sands tailings ponds and oil refineries. We used what is known of similarly sized bioreactors and hydrocarbon recovery techniques to decide what factors to consider in the design of OSCAR's home: culture conditions, method for hydrocarbon extraction, and containment of the genetically modified organisms. <br />
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<h2>Research</h2><br />
</html>[[File:Wastewater plant-ucalgary.JPG|thumb|200px|right|Figure 1: Visiting Calgary's Bonneybrook wastewater treatment plant, overlooking one of the bioreactors. ]]<html><br />
<p>Before diving into making a bioreactor, we first had to research current solutions in the field. To help us with this phase, we read papers on bioreactors that exist with such diverse applications as wastewater treatment, tissue engineering and beer fermentation.<br />
To observe a large scale bioreactor, we toured a wastewater treatment plant (we would have preferred a brewery) where we interviewed plant managers and learned conditions that need to be considered in big systems: open or closed system (theirs was open), methods for oxygenation and preventing contents from settling. We also interviewed graduate students and professors doing research on bioreactors at the University of Calgary for their insight as well as meeting weekly with the supervisors and biologists on our team. Here is a picture from our trip to the wastewater plant!</p><br />
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<h2>Our Bioreactor Evolution</h2><br />
<p>Throughout the summer we worked on creating a prototype of the bioreactor. The process that was deemed most suitable was a cross between a fed-batch system and a continuous stir method in a closed system, where the reactors would be continually fed with more bacterial nutrients and fresh tailings. To remove the hydrocarbons from the culture we decided to use a belt skimmer, similar to those used to help clean up oil spills. This method allows us to run the belt to pick up hydrocarbons without having to remove the entire solution of the batch. This way the bacterial culture already present in the tank can be maintained in active culture to continuously produce more hydrocarbons, which is favored for an industrial scale (1000+ L tanks). Tailings are pre-filtered to prevent environmental strains from joining the mix. Additionally, the process would have to occur within an enclosed system to ensure its containment.</p> <br />
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<p>To make sure that the belt does not transfer live bacteria into the hydrocarbon collection tank, we will have a UV light aimed at the most apical point in the belt path to ensure that any bacteria picked up by the skimmer receive a lethal dose of radiation just before the hydrocarbons are removed from the bioreactor chamber.</p><br />
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</html>[[File:UCalgary2012_BioreactorOverview.jpg|thumb|745px|left|Figure 2: From computer to prototype: how we made our bioreactor. a) Our system began with a model built using Google Sketch Up. It had two chambers and a tube acting as a siphon to pull off hydrocarbons. b/c) The first prototype took shape using materials we found in the lab (including the recycling bin). This system was meant to show that the bioreactor could agitate a solution with the turbine. d) This prototype is the first manufactured design we put together. It needs a power source to turn on the computer fan motor which runs the turbine. It also includes an air sparger system to allow our system to be oxygenated. Apart from the plastic gears, it is fully autoclavable. e/f) Our final prototype, which includes the belt skimmer in an enclosed system. It is able to skim off the top oil layer in a solution of water and canola oil into a small falcon tube.]]<html><br />
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<h2>The Prototype Design</h2><br />
<p>We determined the essential concepts that needed to be developed in the prototype. As with the scaled up design, we included the belt skimmer, powered by a small motor to move the belt into and out of the system. Since our bioreactor would necessarily have live cells, our prototype operated as a completely closed system to prevent cross contamination with microbes outside the chamber. </p><br />
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<h2>The Belt Skimmer in Action</h2><br />
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<p> This video showcase our belt skimmer. The model hydrocarbons stick to the belt shown in the video and are skimmed off into a Tim Horton's coffee cup (the only disposable cup we could find in the lab. </p><br />
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<h2>Current Prototype with both the Sparger and Turbine</h2><br />
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<p> The second video shows our bioreactor prototype in action with both the sparger and turbine running. </p><br />
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<h2>Belt Skimmer with a Collection Chamber</h2><br />
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<p> This video shows the collection chamber we added to the bioreactor. There is a hole at the bottom of the chamber so that during the bioreactors' showcase the model hydrocarbons don't accumulate in the chamber. </p><br />
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<br />
<p>Once we had these designs in place, we were able to start building models and presentation material. One of our goals was to have a computer animation of our design in motion. We were able to meet this goal by using the Maya and RealFlow programs. Maya is a complex and extremely versatile computer animation program used in many animated movies, including James Cameron’s “Avatar”. RealFlow is a particle-generating program, used primarily for creating fluid flow and fluid effects. Combining both of these programs, we created a seventeen second long video showing the basic idea of how our bioreactor will work. Our model will be brought to the competition for demonstration purposes.</p><br />
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<h2>Particle Simulation Using RealFlow2012</h2><br />
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<h2>Open System Showing Separation of Hydrocarbon Layer</h2><br />
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<h2>Closed System Showing Emulsified Hydrocarbons</h2><br />
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<h2>Testing and Results</h2><br />
<p>Using the physical models that we made, we were able to conduct experiments to help determine what will make our design most efficient. We received five different belt samples from a belt skimming company (Abanaki), and conducted three different tests to determine which belt is most suitable for us. Our tests sought to find the belt that picked up the most oil, least tailing pond material, and least amount of bacteria. </p><br />
</html>[[File:UCalgary-Bioreactor-Materials.jpg|thumb|745px|left|Figure 3: Left panel: Belt rankings from three different tests. We tested the ability of the belts to pick up hydrocarbons, and to exclude bacteria or tailings pond water. Right panel: The five belts we tested and a sample of canola oil used for hydrocarbon pick up. From left to right: metallic material, blue texture, fur belt, white plastic, white texture]]<html><br />
<br />
<p>Additionally, we ran three different twenty-four hour bacterial growth tests in our tank to determine the effectiveness of the agitator and sparger on bacterial growth. The turbine mixes the solution so as to prevent the settling of bacterial cells and other heavier materials and to ensure even nutrient and reactant distribution in the tank. The sparger aerates the solution, which is necessary for aerobic bacteria to thrive. When assembled together, the turbine is located above the sparger thus breaking each bubble from the sparger into smaller ones. The test was conducted with a turbine and a sparger, a turbine only, and a sparger only. At the end of each experiment we measured the optical density of the solution with a spectrophotometer to quantify the bacterial growth. Operating our bioreactor with both a turbine and sparger resulted in slightly greater bacterial growth than just the turbine, which coincides with our hypothesis. In order to use the air sparger, we decided to use a HEPA filter to screen air coming out of the system to maintain constant pressure in the tank. The results are displayed below:</p><br />
<br />
</html>[[Image:UCalgary-Bioreactor-ODNA.jpg|thumb|745px|left|Figure 4: This is the spinner flask and sparger system we used for our bacterial growth tests. Each bacterial growth experiment lasted 24 hours in an incubator at 37 degrees Celsius. This is our data for the optical density reading of each bacterial growth experiment. As expected, we had the most growth when both the turbine and sparger were in operation for 24 hours.This image shows NA and Hydrocarbon Separation in a falcon tube after sitting for 5 minutes. As can be seen, a hydrocarbon layer forms on top of the naphthenic acid layer. This was a very encouraging result since we want to skim the hydrocarbon products from the top layer of our bioreactor.]]<html><br />
<br />
<p> Furthermore, we tested our belts' ability to pick up hydrocarbons in a solution of water and commercial naphthenic acid. We dipped our belt in a solution of hexadecane, water and naphthenic acid, then removed and scraped the picked up solution into a separate beaker. This sample was then run through GC-MS to analyze the concentration of naphthenic acid found in our skimmed solution. This is an important test since we do not want to be removing too many NA's before they have the chance to be converted to hydrocarbons. The results of the GC-MS were very promising. Since we used commercial NA's, many different types of NA's were represented in our original solution. To find the concentration of each NA, the number of carbon rings for each type of NA are counted. As can be seen below, the figures show plots of the number of carbon rings of each NA found in the water layer and hydrocarbon layer of our skimmed solution. Based on the size of the bars on the graph, our data shows that a higher abundance of NA were found to be associated with the water and not the hydrocarbon layer. This data suggests that minimal NA's were found in our skimmed hydrocarbon layer and that most were left in the water layer. <br />
</html>[[File:HC layer, skimmed (no NaOH)-ucalgary.png|thumb|600px|center|Figure 5: This graph shows the amount of NA's found in the skimmed hydrocarbon layer. Each bar represents the carbon ring count for a different type of NA. Clearly, minimal NA's were skimmed into the hydrocarbon layer.]]<html><br />
</html>[[File:Water layer, skimmed-ucalgary.png|thumb|600px|centre|Figure 6: This graph shows how many NA's were left in the water layer of our skimmed solution. This data suggests that minimal amounts of NA were skimmed into our solution, and with most of those skimmed found in the water layer.]] <html><br />
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<br />
<h2>The Final System</h2><br />
<p>Along with physical considerations of the containment unit, we must also consider the composition and growth of the bacteria in the reactor. Each OSCAR bacterium would have the most suitable kill-switch circuit attached to its respective hydrocarbon conversion circuit. The bacterium would also have a deletion of a gene for the biosynthesis of glycine, which would be supplemented in our defined production media. We envision OSCAR to be a co-culture of decarboxylation, decatecholization, denitrogenation, and desulfurization. Lastly, due to the energetically expensive nature of maintaining the circuits, we anticipate that if the circuits are constitutively produced cell growth rate may be very slow. Therefore in the final circuits we may want them to be activated by quorum sensing promoter systems. Essentially, when cells are at low density they focus energy on growth; when cells reach appropriate density for the reaction chamber, transcription of the circuit is enabled. Together we hope that the system will clean up recalcitrant petroleum waste and produce energy.<br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-26T09:30:24Z<p>MaggieRY: </p>
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<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team began our project with human practices in mind. While we had established a research objective to produce a biosensor and bioreactor system, we wanted to ensure that our system was relevant to the industry where it would be employed. As well, we wanted to ensure that academic, government, and industry professionals' concerns were taken into consideration during the design process of our system. In order to best accomplish this, we conducted interviews with two leaders in oilsands reclamation. We approached a major oilsands company, Suncor, and talked to Christine Daly, an Ecologist who works in Environmental Cleanup. We then approached Ryan Radke, the president of BioAlberta. BioAlberta focuses on bringing biotechnology to our province and develop these in an industrial setting. His experience allowed us to better predict if our project would have any concerns amongst legislators and industrial leaders. <br />
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<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
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<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
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<h2>Follow-Up Interviews</h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
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<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
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}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-26T09:24:17Z<p>MaggieRY: </p>
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<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
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<br><h2>Why choose hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
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<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of disrupting metabolism or the limitations of a single channel output.</p><br />
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<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
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[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
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<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
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<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
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<h2>Genes, Chemicals, and Circuits</h2><br />
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<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
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[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
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<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
<br />
<h2>Does it Work?</h2><br />
<br />
<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
<br />
</html><br />
[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
<html><br />
<br />
<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
<br />
<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
<br />
<h2>What Next?</h2><br />
<br />
<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
<br />
</html>}}}<br />
<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-26T09:03:12Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Novel Electrochemical Reporting System|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_FRED_Reporting_Low-Res.png" style="padding: 10px; width: 225; float: right;"></img><br />
<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
<br />
<br><h2>Why choose hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
<br />
<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of cutting off metabolism or having to use only a single channel output.</p><br />
<br />
<br />
<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
<br />
</html><br />
[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
<html><br />
<br />
<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
<br />
<br><br />
<br />
<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
<br />
<br><br />
<br />
<h2>Genes, Chemicals, and Circuits</h2><br />
<br />
<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
<br />
</html><br />
[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
<html><br />
<br />
<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
<br />
<h2>Does it Work?</h2><br />
<br />
<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
<br />
</html><br />
[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
<html><br />
<br />
<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
<br />
<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
<br />
<h2>What Next?</h2><br />
<br />
<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
<br />
</html>}}}<br />
<br />
}}</div>MaggieRYhttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-26T07:38:45Z<p>MaggieRY: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Novel Electrochemical Reporting System|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_FRED_Reporting_Low-Res.png" style="padding: 10px; width: 225; float: right;"></img><br />
<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
<br />
<br><h2>What's the big idea?</h2><br />
<p>To get our bacteria to report on the toxic compounds present in the tailings ponds we needed a quick and reliable system that would function in a wide variety of environments, including murky solutions. We turned to electrochemistry for this, as the turbidity of the solution doesn't effect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product or to transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are through the action of metal respiration, phosphatases, or hydrolases. </p><br />
<br />
<p>The first system, that of the respiration of metals, involves using an organism that uses ions such as Fe<sup>3+</sup> as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it would require throttling the entire metabolic network of an organism such that it could only produce the electric signal, and also breathe, under a very specific condition. It is also limited to a single output,, as although these bacteria can usually respire a few different kinds of metals, they use the same pathway to do this, meaning that there would be no way for the bacterium to discern between the two different signals it could produce.</p><br />
<p>The second system relies on phosphatases to remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of cell viability, it also is limited to a single output, as the non-specific phosphatases would act on multiple substrates in a solution. Another issue that could reduce the effectiveness of this system is that there would be background expression of phosphatases in the bacterium as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we turned to a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of metabolism throttling or having to use only a single channel output.</p><br />
<br />
<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for it's electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
<br />
</html><br />
[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
<html><br />
<br />
<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of it's fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
<br />
<br><br />
<br />
<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
<br />
<br><br />
<br />
<h2>Genes, Chemicals, and Circuits</h2><br />
<br />
<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
<br />
</html><br />
[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
<html><br />
<br />
<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. As the <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry has a frameshift mutation rendering the enzyme nonfunctional, one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a> was used for initial characterization. The <i>bglX</i> and <i>uidA</i> genes were amplified from the <i>E. coli</i> genome using PCR and biobricked as <a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a> and <a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a> respectively. These genes were then constructed under the <a href="http://partsregistry.org/Part:BBa_R0010"><i>lacI</i> promoter</a> to allow for comparison testing.</p><br />
<br />
<h2>Does it Work?</h2><br />
<br />
<p>Yes! We have been able to show that we can detect the action of our hydrolase enzymes acting on the sugar-conjugated compounds to give us an electrochemical signal (<b>Figure 4</b>).</p><br><br />
<br />
</html><br />
[[File:UCalgary2012-Electrochem-Robert.jpg|thumb|700px|center|Figure 4: A) Detection of <i>lacZ</i> activity on CPRG at 1.325V vs RHE through the production of CPR. B) Cleavage of PDPG into PDP by <i>bglX</i> being detected at 0.825V vs RHE. C) The action of <i>uidA</i> on PNPG at 1.6V vs RHE when under the control of the <html><a href="http://partsregistry.org/Part:BBa_R0010">R0010</a></html> promoter induced with IPTG or uninduced.]]<br />
<html><br />
<br />
<p>These graphs show two main points. The first being that we can successfully use hydrolase enzymes as reporters for gene expression with a sensitive output. This gives us the power to accurately watch bacteria respond to a stimuli in real time with the ability to differentiate between minute differences in expression strength. As these reporters do not rely on having a colour or fluorescence output they can be used in turbid solutions and even solutions free from oxygen. This removes two of the major limitations of current biosensors, allowing this branch of biotechnology to access a broad new market.</p><br />
<br />
<p>The second interesting conclusion that can be drawn for part C of Figure 4 is the leakiness of the <a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a> promoter. The bacteria were induced at time zero and a clear increase is seen almost immediately for the induced trial, but the current does still increase over time for the uninduced test. The leaky expression of the genes downstream of this promoter could be detrimental in situations such as toxic gene expression or time dependent events.</p><br />
<br />
<h2>What Next?</h2><br />
<br />
<p>With our electrochemical system functioning properly we can now hook up our reporter genes to promoters found in the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon library</a> for a final detection system. We have also created a <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">hardware and software platform</a> for a field-ready biosensor. Our system has also been <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">mathematically modeled</a> in MATLAB to aid us in planning time courses for the experiments and the final prototype. When combined with the mechanical and biological containment mechanisms used in our system these genes create a novel and safe approach to biosensing in the oil sands and in many other potential applications.</p><br />
<br />
</html>}}}<br />
<br />
}}</div>MaggieRY