http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Emily+Hicks&year=&month=2012.igem.org - User contributions [en]2024-03-29T01:20:13ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Project/AttributionsTeam:Calgary/Project/Attributions2013-10-23T05:43:09Z<p>Emily Hicks: Undo revision 301439 by Himika9 (talk)</p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Attributions|<br />
CONTENT=<br />
<br />
<html><br />
<h2>University of Calgary Support</h2><br />
<p>The University of Calgary iGEM team would like to thank and acknowledge all of the support from the University in terms of student stipends, support personnel, facilities, and materials which made this project possible. The <b>Department of Biological Sciences </b> and the <b>O'Brien Centre for the Bachelor of Health Sciences </b> provided the laboratories we worked in this summer, and the O'Brien Centre allowed us to continue our work even into the fall term. Additionally, a special thanks to all departments and faculties which financially contributed to our project.</p><br />
<br />
<h2>Advisor and Instructor Support</h2><br />
<p>All research work was performed by the undergraduate students on our team. Our professors and advisors contributed support and ideas to the students and facilitated a safe and efficient laboratory environment. Special thanks to <b>Dr. Lisa Gieg</b>, whose experience in petroleum microbiology provided the facilities and protocols required to make our work possible (including GC/MS and culturing of various organisms). And thanks to <b>Dr. Anders Nygren</b> for his help with engineering the electrical circuits used in the our biosensor prototypes. Finally, thanks to <b>Dr. Mayi Arcellana-Panlilio</b> for her troubleshooting advice.</p><br />
<br />
<h2>Support From Additional Professors and University Staff</h2><br />
<p>Special thanks to <b>Dr. Doug Muench</b>, one of our advisors whose support made it possible for us to obtain our laboratory space in Biological Sciences. In addition, we would like to acknowledge other professors in the department of Biological Sciences including <b>Dr. Greg Moorhead</b>, <b>Dr. C. C. Chinnappa</b> (both for supplying equipment), <b>Dr. Michael Hynes</b>, <b>Dr. Ray Turner</b>, <b>Dr. Gerrit Voordouw</b>, <b>Dr. Howard Ceri</b>, <b>Dr. Denice Bay</b>, and <b>Monika Schwering</b> for their support with supplies and protocols. Additionally we would like to recognize <b>Dr. Viola Birss</b>, <b>Bri Campbell</b>, and <b>Anusha Abhayawardhana</b> in the Department of Chemistry for their assistance with the electrochemical studies conducted by our team. Finally we would like to thank <b>Dr. Arin Sen</b> in the Department of Chemical and Petroleum Engineering for his advice with the design of our bioreactor and <b>Dr. Jennifer Cobb</b>, <b>Deirdre Lobb</b>, <b>Dr. Steve Robbins</b>, and the <b>SACRI Research Group</b> for their donation of chemicals. </p> <br />
<br />
<h2><i>Outside of University</i> Research and Technical Support</h2><br />
<p>We would like to thank and acknowledge the support of various individuals from other Universities. This includes<br />
<b>Dr. Michael Ellison</b> for his lab's contribution of Keio Collection Knockout strains used in this project, the <b>2011 University of Washington iGEM team</b> for sending us plasmid DNA of the Petrobrick as the DNA construct available in the registry was incorrect. Thanks to <b>Dr. Josie L. Garcia</b> from the Consejo Superior de Investigaciones, Madrid Spain for contribution of a plasmid containg the <i>hpaC</i> gene. <b>Dr. John Kilbane</b> for the contribution of the <i>Rhodococcus baikonurensis</i> containing the <i>Rhodococcus</i> IGTS8 <i>dszABC</i> plasmid. Special thanks to our professor <b>Dr. Lisa Gieg</b> for her contribution of a <i>Pseudomonas sp.</i> LD2 species previously reported to degrade carbazole. Finally, special thanks to <b>Abanacai Corporation</b>, Ohio for their samples of a oil skimming sample kit used in our bioreactor design. </p><br />
<br />
<h2><i>Outside of University</i> Additional Support</h2><br />
<p>We would like to thank the individuals who we had requested to do interviews with <b>Christine Daly</b> (Suncor Inc.), <b>Ryan Radke</b> (BioAlberta), <b>Kelly Roberge</b> (K Roberge Consulting Ltd.), <b>William Sawchuk</b> (ARC Resources), <b>Gordon Lambert</b> (Suncor Inc.), and <b>Zvonko Burkus</b> (Alberta Environment). We would like to thank our representative <b>Claudia Bustos</b> for all of her hard work and support of our team from the Telus Spark Science Centre. Thank you to <b>Robert Kotch</b> from the Bonniebrook Waste Treatment Plant for allowing us access to their facility to learn more about how their reactors were designed. We would also like to thank <b>Lorne and Laurie Swalm</b> for their generous support of our project. Finally, we would like to thank the E. coli Genomic Stock Centre at Yale University for their support and quick release of the <i>glyA</i> Keio Knockout gene.</p> <br />
<br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-27T03:57:12Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Novel Electrochemical Reporting System|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_FRED_Reporting_Low-Res.png" style="padding: 10px; width: 225; float: right;"></img><br />
<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
<br />
<br><h2>Why Choose Hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
<br />
<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of disrupting metabolism or the limitations of a single channel output.</p><br />
<br />
<br />
<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for its electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
<br />
</html><br />
[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
<html><br />
<br />
<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of its fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
<br />
<br><br />
<br />
<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
<br />
<br><br />
<br />
<h2>Genes, Chemicals, and Circuits</h2><br />
<br />
<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
<br />
</html><br />
[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
<html><br />
<br />
<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. <br />
<br />
There is a <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry already registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>). Upon testing this circuit however, it was determined to not be functional. In a simple assay where we added CPRG to cells expressing this part and induced with 200 mM IPTG, we saw no color change, as we would have expected if the LacZ enzyme was being produced and cleaving the CPRG (yellow) into CPR (red). This can be seen below</p> <p></html>[[File:UofC LacZ assay data.jpg|300px]][[File:UofC_LacZ2!.jpg|200px]]<html></p>.<br />
<br />
<p> Sequencing of this part showed that it had a frameshift mutation. Original validation of our system was thus done using one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a>. After Regionals, a new circuit was constructed and submitted as <a href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>. A similar assay was performed and the results can be seen above as well. On the right is the new circuit, while on the left, the old circuit. The red color indicates that the new pat is indeed functional. This circuit was used in later testing using the LacZ enzyme. 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 />
</html><br />
[[File:CALGARYROBERTOMGTHISPICROCKS.png|thumb|600px|center|Figure 5: Current production from the <i>lacZ</i> and <i>uidA</i> systems under the IPTG inducible promoter. Both samples were run with the same conditions and held at the oxidation potentials of their respective analytes.]]<br />
<html><br />
<br />
<p>We have also been able to directly compare the <i>lacZ</i> and <i>uidA</i> circuits under the same promoter, as shown in Figure 5. In doing this we see twice the current in the <i>uidA</i> system as opposed to the <i>lacZ</i> system with the same conditions. This is because the analyte produced through the action of <i>uidA</i> produces a product that oxidizes to release two electrons while the <i>lacZ</i> product only releases one electron when oxidized. As the current we measure is this release of electrons, a similar amount of the two enzymes would results in the doubling of current for <i>uidA</i> that we saw, showing that our systems are working as expected.</p><br />
<br />
<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>Emily Hickshttp://2012.igem.org/File:UofC_LacZ2!.jpgFile:UofC LacZ2!.jpg2012-10-27T03:55:22Z<p>Emily Hicks: </p>
<hr />
<div></div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-27T03:52:14Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Novel Electrochemical Reporting System|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_FRED_Reporting_Low-Res.png" style="padding: 10px; width: 225; float: right;"></img><br />
<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
<br />
<br><h2>Why Choose Hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
<br />
<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of disrupting metabolism or the limitations of a single channel output.</p><br />
<br />
<br />
<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for its electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
<br />
</html><br />
[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
<html><br />
<br />
<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of its fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
<br />
<br><br />
<br />
<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
<br />
<br><br />
<br />
<h2>Genes, Chemicals, and Circuits</h2><br />
<br />
<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
<br />
</html><br />
[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
<html><br />
<br />
<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. <br />
<br />
There is a <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry already registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>). Upon testing this circuit however, it was determined to not be functional. In a simple assay where we added CPRG to cells expressing this part and induced with 200 mM IPTG, we saw no color change, as we would have expected if the LacZ enzyme was being produced and cleaving the CPRG (yellow) into CPR (red). This can be seen below</p> <p></html>[[File:UofC LacZ assay data.jpg|200px]]<html></p>.<br />
<br />
<p> Sequencing of this part showed that it had a frameshift mutation. Original validation of our system was thus done using one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a>. After Regionals, a new circuit was constructed and submitted as <a href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>. A similar assay was performed and the results can be seen above as well. 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 />
</html><br />
[[File:CALGARYROBERTOMGTHISPICROCKS.png|thumb|600px|center|Figure 5: Current production from the <i>lacZ</i> and <i>uidA</i> systems under the IPTG inducible promoter. Both samples were run with the same conditions and held at the oxidation potentials of their respective analytes.]]<br />
<html><br />
<br />
<p>We have also been able to directly compare the <i>lacZ</i> and <i>uidA</i> circuits under the same promoter, as shown in Figure 5. In doing this we see twice the current in the <i>uidA</i> system as opposed to the <i>lacZ</i> system with the same conditions. This is because the analyte produced through the action of <i>uidA</i> produces a product that oxidizes to release two electrons while the <i>lacZ</i> product only releases one electron when oxidized. As the current we measure is this release of electrons, a similar amount of the two enzymes would results in the doubling of current for <i>uidA</i> that we saw, showing that our systems are working as expected.</p><br />
<br />
<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>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/FRED/ReportingTeam:Calgary/Project/FRED/Reporting2012-10-27T03:49:14Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectGreen|<br />
TITLE=A Novel Electrochemical Reporting System|<br />
<br />
CONTENT={{{CONTENT|<br />
<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/UCalgary2012_FRED_Reporting_Low-Res.png" style="padding: 10px; width: 225; float: right;"></img><br />
<p>For FRED to be able to tell us about the toxins he's sensing we needed a good reporter system that could function in a wide array of environments. Unfortunately the traditional fluorescent or luminescent reporters have significant drawbacks that prevent them from being useful in a tailings environment that is murky and potentially anaerobic. Due to these limitations we decided to improve upon <a href="https://2011.igem.org/Team:Calgary">last year's single output electrochemical sensor</a> using the <i>lacZ</i> gene to cleave a substrate into an easily detectable analyte. Our team has developed a novel system that utilizes <b>three separate reporter genes</b> to provide a triple-output electrochemical biosensor and can be used in a wide variety of applications. This system overcomes traditional reporters in that it is <b>fast</b>,<b> accurate</b>, and can <b>function in turbid environments</b> and even in the <b>absence of oxygen!</b></p><br />
<br />
<br><h2>Why Choose Hydrolases?</h2><br />
<p>To get our bacterial biosensors to report toxic compounds present in the tailings ponds, we needed a quick and reliable system that would function in a variety of aqueous environments. We turned to electrochemistry for this, as the turbidity of the solution doesn't affect the results and nanomolar levels of chemicals can consistently be detected. The idea behind electrochemistry is that the bacteria would either cleave a substrate to produce an oxidizable product (analyte), or transfer electrons directly into an electrode. The three most common methods through which bacteria produce an electrical response are the activities of phosphatases, hydrolases, and metal respiration. </p><br />
<br />
<p>The first system, that of the respiration of metals, involves using an organism that uses metal ions, such as Fe<sup>3+</sup>, as the terminal electron acceptors in the cellular respiration pathways. While this kind of a system has the potential to be useful in creating bioelectricity, its use as a biosensor is limited. This is because it requires putting one of the essential electron transport genes under an inducible promoter, such that when the promoter is activated, respiration is enabled causing a change in current. Although these bacteria can usually respire more than one type of metal, they bottleneck to a single pathway and output.</p><br />
<p>The second system relies on phosphatases: enzymes that remove a phosphate group from an electrochemical analyte. When the phosphate group is removed the resultant product could be oxidized or reduced at an electrode to produce a response that would be measured as a change in current. While this method solves the problem of reduced cell viability created in the first system, it also is limited to a single output, as the non-specific phosphatases would act on all substrates in a solution. The effectiveness of the system could be further reduced by background expression of phosphatases in the bacterium, as these enzymes are essential for processes such as signalling and metabolism. </p><br />
<p>With this in mind we favoured a hydrolase based system, which offers the versatility and sensitivity of electrochemistry, without the pitfalls of disrupting metabolism or the limitations of a single channel output.</p><br />
<br />
<br />
<br><h2>How Does it Work?</h2><br />
<a name="hydrolase"></a><p>The enzymes encoded by our reporter genes are specific sugar hydrolases. This means that they target one kind of sugar and remove it from whatever compound they are attached to. We have chosen to use the sugars glucose, glucuronide, and galactose for our system. The genes responsible for their respective hydrolases are <i>bglX</i> (<a href="http://partsregistry.org/Part:BBa_K902004">BBa_K902004</a>), <i>uidA</i> (<a href="http://partsregistry.org/Part:BBa_K902000">BBa_K902000</a>), and <i>lacZ</i> (<a href="http://partsregistry.org/Part:BBa_I732005">BBa_I732005</a>). By having our electrochemical analyte conjugated to this sugar, when the hydrolase is expressed the sugar is cleaved from the analyte, allowing for its electrochemical detection. A diagrammatic representation of this system is shown below in Figure 1.</p><br />
<br />
</html><br />
[[File:Calgary2012 EchemWikiFig1.jpg|thumb|600px|center|Figure 1: Representation of cleavage of the sugar-analyte substrate by a hydrolase enzyme.]]<br />
<html><br />
<br />
<p>After the analyte is released we need to detect it. Electrochemistry is an excellent approach for this because of its fast and quantitative nature. A voltage is applied between two electrodes compared to a reference electrode and the resulting current is measured. By changing the applied voltage to that of the oxidation voltage of one of our analytes, the increase in current due to its oxidation when compared to an analyte free baseline is proportional to the amount of analyte present in the solution. This process happens so quickly that you can have an output value in a matter of seconds.</p><br />
<br />
<br><br />
<br />
<p>We used two different electrochemical techniques in our testing depending on what question the experiment was trying to answer. When we were characterizing the voltages at which our products oxidized we used <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">cyclic voltammetry</a>, which is where you apply a voltage and then slowly increase and decrease it over a designated sweep range. Any bumps in the graph are due to a reaction and can be standardized against baseline measurements. After the oxidation potential has been localized we can speed up our experiments by using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">potentiostatic runs</a>. In this case, instead of sweeping the voltage we apply to the solution we hold it steady at the voltage that will oxidize our compound the moment it is released into the solution. Both of these techniques require the three electrodes in an electrolyte solution such as phosphate buffered saline and can routinely detect nanomolar concentrations of electrochemical analytes.</p><br />
<br />
<br><br />
<br />
<h2>Genes, Chemicals, and Circuits</h2><br />
<br />
<p>For our system to have a triple output we need three separate genetic circuits with three analytes possessing unique oxidation potentials. If one chemical overlaps with another we could get false-positives of one chemical due to oxidation of another. To this end we have chosen to use chlorophenol red (CPR), para-diphenol (PDP), and para-nitrophenol (PNP). These compounds are conjugated with their sugars to form CPR-&beta;-D-galactopyranoside (CPRG), PDP-&beta;-D-glucopyranoside (PDPG), and PNP-&beta;-D-glucuronide (PNPG). An easy way to tell the analytes from their sugar conjugates is the addition of the letter G to the acronym. These chemicals are summarized below in Figure 2 along with the reporter genes used with each one.</p><br />
<br />
</html><br />
[[File:Calgary2012 ECHEMWikiFig2.png|thumb|700px|center|Figure 2: Analyte/sugar combinations as well as the reporter genes responsible for the detection of each compound.]]<br />
<html><br />
<br />
<a name="output"></a><p>Out of the three sugar conjugates the only one that exhibits any electrochemical activity is PDPG, with it's oxidation potential at 0.6V vs. the reduction of hydrogen reference electrode (RHE). The three analytes have potentials at 0.825V for PDP, 1.325V for CPR, and 1.6V for PNP vs RHE. As none of these peaks overlap and no sugar conjugates interfere with their signals the three chemicals can be detected in the same solution. Figure 3 shows sensitive simultaneous detection of our three analytes with no background interference.</p><br />
<br />
</html><br />
[[File:Calgary2012 FRED triple.png|thumb|500px|center|Figure 3: Cyclic voltammogram of the three electrochemical analytes vs RHE. PDP has a peak at 0.825V, while CPR is at 1.325V and PNP is at 1.6V. The concentration of all analytes was 40&micro;M]]<br />
<html><br />
<br />
<p>With the chemicals finalized we now needed to construct our circuits. <br />
<br />
There is a <i>lacZ</i> gene under the control of the <i>lacI</i> promoter in the registry already registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>). Upon testing this circuit however, it was determined to not be functional. In a simple assay where we added CPRG to cells expressing this part and induced with 200 mM IPTG, we saw no color change, as we would have expected if the LacZ enzyme was being produced and cleaving the CPRG (yellow) into CPR (red). This can be seen below </html>[[File:UofC LacZ assay data.jpg]]<html>.<br />
<br />
<p> Sequencing of this part showed that it had a frameshift mutation. Original validation of our system was thus done using one of the constitutive <i>lacZ</i> hits from the <a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">transposon screen</a>. 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 />
</html><br />
[[File:CALGARYROBERTOMGTHISPICROCKS.png|thumb|600px|center|Figure 5: Current production from the <i>lacZ</i> and <i>uidA</i> systems under the IPTG inducible promoter. Both samples were run with the same conditions and held at the oxidation potentials of their respective analytes.]]<br />
<html><br />
<br />
<p>We have also been able to directly compare the <i>lacZ</i> and <i>uidA</i> circuits under the same promoter, as shown in Figure 5. In doing this we see twice the current in the <i>uidA</i> system as opposed to the <i>lacZ</i> system with the same conditions. This is because the analyte produced through the action of <i>uidA</i> produces a product that oxidizes to release two electrons while the <i>lacZ</i> product only releases one electron when oxidized. As the current we measure is this release of electrons, a similar amount of the two enzymes would results in the doubling of current for <i>uidA</i> that we saw, showing that our systems are working as expected.</p><br />
<br />
<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>Emily Hickshttp://2012.igem.org/File:UofC_LacZ_assay_data.jpgFile:UofC LacZ assay data.jpg2012-10-27T03:30:52Z<p>Emily Hicks: </p>
<hr />
<div></div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:26:59Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’ve 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 some of these parts, and how do they work together? Our next major goal was therefore to <u><b>establish synergy:</b> try to put these pieces together in order to assess how far we’d 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. All our electrochemical protocols can be found here. 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 />
<br />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared 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 t 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 />
<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 />
<br />
<h2><u>Putting OSCAR together </u></h2><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 />
<b> Insert video here</b><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 />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:18:25Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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 <u><b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d 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 transposon we have identified. 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. All our electrochemical protocols can be found here. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|thumb|550px|centre|Figure 3. ]]<html><br />
<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 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 t 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 />
<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 />
<br />
<h2><u>Putting OSCAR together </u></h2><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 />
<b> Insert video here</b><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 />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-27T03:16:06Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
<br />
/*link coloring schema*/<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
a.purple{<br />
color: #C5B !important;<br />
}<br />
<br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/3/3d/UCalgary2012_FRED_and_OSCAR_Achievements.png" style="width: 280px; float: right; padding: 10px;"></img><br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews"><b>Returned to industry experts</b></a> to see if we accomplished our goals and what the <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>next steps</b></a> should be.</li></p><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><b>Characterized</b></a> the functionality of our previously submitted <a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066"><b><i>Prha</i> (BBa_K902066)</b></a> promoter using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><b>complete kill switch device</b></a> using the <a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084"><b><i>Prha</i> (BBa_K902084)</b></a> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation"><b>Further characterized</b></a> our previously validated <b>magnesium riboswitch kill gene construct</b><a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018"> <b>(BBa_K902018)</b></a>.</li></p></ul><br />
<br />
<br><br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting"><b>Electrochemically characterized</b></a> an inducible <a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902090"><b><i>lacz</i> (BBa_K902090)</b></a> circuit, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting"><b>Electrochemically characterized</b></a> a constitutive <a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902005"><b><i>bglX</i> (BBa_K902005)</b></a> <b>generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Tested one of our transposon library hits</b></a> with our electrochemical reporter, showing that we can use our system to <b>detect toxins electrochemically</b>.</p></li><br />
<br />
<li><p><a class="green" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Validated our system with tailings</b></a> using our transposon system and electrochemical reporter to be able to selectively detect toxins on <b>real oil sands samples</b>.</p></li><br />
</ul><br />
<br />
<br><br />
<br />
<p><b>In terms of <FONT COLOR=#1088CC>OSCAR</FONT>, we...</b></p><br />
<ul><br />
<li><p>Obtained <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>additional characterization data</b></a> in validation of our flux-variability analysis model.</p></li><br />
<br />
<li><p>Characterized the ability of our <b>novel</b> <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041"><b><i>amdA</i> (BBa_K902041)</b></a> <b>BioBrick</b> to selectively remove primary amides from ring structures with remarkable efficiency, turning them into a substrate that the <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025"><b>Petrobrick (BBa_K590025)</b></a> can likely convert into hydrocarbons.</p></li><br />
<br />
<br />
<li><p><a class="blue" href="https://2012.igem.org/Team:Calgary/Project/Synergy"><b>Validated a scale up model</b></a> for using our bioreactor/belt skimmer system in producing and extracting hydrocarbons.</p></li><br />
</ul><br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:11:03Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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 <u><b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d 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 transposon we have identified. 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. All our electrochemical protocols can be found here. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png|550px|centre|Figure 3. ]]<html><br />
<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 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 t 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 />
<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 />
<br />
<h2><u>Putting OSCAR together </u></h2><br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</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.</p><br />
<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 />
<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 />
<b> Insert video here</b><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 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 />
<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 />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:08:50Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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 <u><b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d 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 transposon we have identified. 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. All our electrochemical protocols can be found here. The results of this assay can be shown below.</p><br />
<br />
</html>[[File:UOFCTailingsPondWinData!.png]]<html><br />
<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 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 t 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 />
<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 />
<br />
<h2><u>Putting OSCAR together </u></h2><br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</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.</p><br />
<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 />
<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 />
<b> Insert video here</b><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 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 />
<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 />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/File:UOFCTailingsPondWinData!.pngFile:UOFCTailingsPondWinData!.png2012-10-27T03:03:47Z<p>Emily Hicks: </p>
<hr />
<div></div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:56:37Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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 <u><b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d 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 transposon we have identified. 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 X: 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 />
<b>INSERT BEAUTIFUL FIGURE HERE!!!!</b><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<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. All our electrochemical protocols can be found here. The results of this assay can be shown below.</p><br />
<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 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 t 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 />
<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 />
<br />
<h2><u>Putting OSCAR together </u></h2><br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</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.</p><br />
<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 />
<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 />
<b> Insert video here</b><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 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 />
<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 />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:50:36Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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 <u><b>establish synergy:</b> try to put some of these pieces together in order to assess how far we’d 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 transposon we have identified. 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 X: 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 />
<b>INSERT BEAUTIFUL FIGURE HERE!!!!</b><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<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. All our electrochemical protocols can be found here. The results of this assay can be shown below. Here we compare our transposon in tailings water to our transposon in a PBS control. This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared 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 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><u>Putting OSCAR together </u></h2><br />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</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.</p><br />
<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 />
<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 />
<b> Insert video here</b><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 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 />
<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 />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:15:54Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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 />
<br />
<h2><u>Putting our Killswitch Together</u></h2><br />
<br />
<h2>Testing our inducible killswitch with our auxotrophic killswitch</h2><br />
<br />
<h2> <u>Putting FRED together</u> </h2><br />
<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 NAs in media. We added __ of commercial naphthenic acids to the media, and monitored the formation of CPR upon the addition of CPRG. We compared this to a control where we used PBS. The results of this can be seen below. Here we can clearly see that we get a response when we're monitoring NAs compared to our contorl. Although we still see some leaky expression in the control, we see a clear difference between the level of induction that we are getting in our assay run as compared to our control run. This was really exciting as it shows that we can in fact detect NA’s electrochemically! FRED works!<br />
<br />
</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<p>It’s great to be able to sense NAs in media, however the real test is can we do it in tailings ponds water! We ran a similar assay where we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Again, upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production. The results of this assay can be shown below. This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to our 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. This shows that FRED is functional in the application that we designed it for! The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. All our electrochemical protocols can be found here.</p><br />
<br />
<h2><u>Putting OSCAR together </u></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. 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 X: 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 />
<b>INSERT BEAUTIFUL FIGURE HERE!!!!</b><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 <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 />
<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 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 />
<br />
<p>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.</p><br />
<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 />
<br />
<br />
<br />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:12:25Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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> <u>Putting FRED together</u> </h2><br />
<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 NAs in media. We added __ of commercial naphthenic acids to the media, and monitored the formation of CPR upon the addition of CPRG. We compared this to a control where we used PBS. The results of this can be seen below. Here we can clearly see that we get a response when we're monitoring NAs compared to our contorl. Although we still see some leaky expression in the control, we see a clear difference between the level of induction that we are getting in our assay run as compared to our control run. This was really exciting as it shows that we can in fact detect NA’s electrochemically! FRED works!<br />
<br />
</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<p>It’s great to be able to sense NAs in media, however the real test is can we do it in tailings ponds water! We ran a similar assay where we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Again, upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production. The results of this assay can be shown below. This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to our 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. This shows that FRED is functional in the application that we designed it for! The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. All our electrochemical protocols can be found here.</p><br />
<br />
<h2><u>Putting OSCAR together </u></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. 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 X: 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 />
<b>INSERT BEAUTIFUL FIGURE HERE!!!!</b><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 <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 />
<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 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 />
<br />
<p>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.</p><br />
<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 />
<br />
<br />
<br />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:11:42Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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’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> <u>Putting FRED together</u> </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 NAs in media. We added __ of commercial naphthenic acids to the media, and monitored the formation of CPR upon the addition of CPRG. We compared this to a control where we used PBS. The results of this can be seen below. Here we can clearly see that we get a response when we're monitoring NAs compared to our contorl. Although we still see some leaky expression in the control, we see a clear difference between the level of induction that we are getting in our assay run as compared to our control run. This was really exciting as it shows that we can in fact detect NA’s electrochemically! FRED works!<br />
<br />
</p><br />
<br />
<h2> Can we sense toxins in tailings ponds? </h2><br />
<br />
<p>It’s great to be able to sense NAs in media, however the real test is can we do it in tailings ponds water! We ran a similar assay where we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Again, upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production. The results of this assay can be shown below. This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to our 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. This shows that FRED is functional in the application that we designed it for! The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. All our electrochemical protocols can be found here.</p><br />
<br />
<h2><u>Putting OSCAR together </u></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. 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 X: 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 />
<b>INSERT BEAUTIFUL FIGURE HERE!!!!</b><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 <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 />
<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 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 />
<br />
<p>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.</p><br />
<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 />
<br />
<br />
<br />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/PartsTeam:Calgary/Parts2012-10-27T01:52:47Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/MainHeader| <html><img src="https://static.igem.org/mediawiki/2012/c/c5/UCalgary2012_HeaderLogoOrange.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|parts|<br />
SECTION=Parts|<br />
SIDELIST=<br />
<html><br />
<ul><br />
<li><a href="#parts">Submitted Parts</a></li><br />
</ul><br />
</html>|<br />
<br />
<br />
TITLE=Parts|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
#groupparts{<br />
float: right;<br />
margin-right: 20px;<br />
}<br />
</style><br />
</head><br />
<body><br />
<img src="https://static.igem.org/mediawiki/2012/a/af/UCalgary2012_FRED_and_OSCAR_Parts.png" style="float: right; padding: 10px;"></img><br />
<p>Here is a collection of all the parts we have submitted over the course of this project. Details of each part can be found on their respective pages on both the Registry and this wiki. </p><br />
<h2>Parts of FRED</h2><p><br />
In terms of FRED, we submitted, two novel hydrolase enzymes as well as generators and composite parts for each.</p><br />
<br />
<h2>Parts of OSCAR</h2><p><br />
In terms of OSCAR, we submitted two novel genes for use in decarboxylation, previously submitted <i>xylE</i> and catalase genes with new promoters, novel desulfurization and denitrogenation enzymes and composite parts and finally, a functional oxidoreductase enzyme expression construct as well as a functional amidase expression construct.</p><br />
<br />
<br />
<br />
<h2>Killswitch Parts</h2><br />
<p><br />
In terms of our killswitch project, we submitted three novel riboswitches and their respective promoters, a novel inducible/repressible promoter as well as composite parts with each including five functional circuits with either GFP or the S7 micrococcal nuclease.</p><br />
<br />
<a name="parts"></a><br />
</body><br />
</html><br />
<br />
<groupparts>iGEM012 Calgary</groupparts><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T01:19:56Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team 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 />
<h2> <u>Initial Interviews</u> </h2><br />
<br />
<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
<p>We spoke with Christine Daly, an Aquatic Reclamation Research Coordinator at Suncor Energy Inc. Christine expressed an interest in our <a href="https://2011.igem.org/Team:Calgary">project in 2011</a> and was willing to discuss this year’s project design with us. One major point that was brought up early on in our design was that there is an opportunity for engineered organisms to outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar concern. To address these concerns, we created our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> system, which would physically contain our bacteria, and also a <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">genetic killswitch mechanism</a>. Another interesting point brought up in this discussion was how the oil industry is currently looking into biology as one of many potential alternative methods to remediate the toxic components of tailings ponds and the oil sands in general. Research exists with other systems such as algal bioremediation, but practical implementations of biology in the oil sands appear to be rather few and far between. Oil industries do, however, appear to show an increased interest in biology (and in turn, synthetic biology) as a possible solution to various problems, a sentiment reflected in <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">our dialogue with the Oil Sands Leadership Initiative</a>.</p><br />
<p>The full interview can be viewed below.</p><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/GiM6EIC9XBo" frameborder="0" allowfullscreen></iframe><br />
<br />
</div><br />
<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/86XQ-Kg5fJ4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
<br />
<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both structural and genetic killswitch devices. In the later part of our project, we have also been trying to work on establishing a glycine auxotrophic killswitch to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Similar to Mr. Roberge, another thing Mr. Sawchuk brought up was scale-up. Specifically, he talked about feasibility and cost a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons, which can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/HumanPractices/InterviewsTeam:Calgary/Project/HumanPractices/Interviews2012-10-27T01:09:21Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Interviews|<br />
CONTENT=<br />
<html><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 280px;"></img><br />
<h2>Purpose</h2><br />
<p> This year the Calgary iGEM team 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 />
<h2> <u>Initial Interviews</u> </h2><br />
<br />
<h3>Talking with Suncor's Christine Daly on Biology in the Oil Sands</h3><br />
<p>We spoke with Christine Daly, an Aquatic Reclamation Research Coordinator at Suncor Energy Inc. Christine expressed an interest in our <a href="https://2011.igem.org/Team:Calgary">project in 2011</a> and was willing to discuss this year’s project design with us. One major point that was brought up early on in our design was that there is an opportunity for engineered organisms to outcompete existing tailings ponds bacteria, and we were pleased to hear that Christine had a similar concern. To address these concerns, we created our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">bioreactor</a> system, which would physically contain our bacteria, and also a <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">genetic killswitch mechanism</a>. Another interesting point brought up in this discussion was how the oil industry is currently looking into biology as one of many potential alternative methods to remediate the toxic components of tailings ponds and the oil sands in general. Research exists with other systems such as algal bioremediation, but practical implementations of biology in the oil sands appear to be rather few and far between. Oil industries do, however, appear to show an increased interest in biology (and in turn, synthetic biology) as a possible solution to various problems, a sentiment reflected in <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">our dialogue with the Oil Sands Leadership Initiative</a>.</p><br />
<p>The full interview can be viewed below.</p><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/GiM6EIC9XBo" frameborder="0" allowfullscreen></iframe><br />
<br />
</div><br />
<br />
<h3>BioAlberta's Ryan Radke on Biology in the Oil Sands</h3><br />
<div align="center"><br />
<iframe width="600" height="450" align="center" src="http://www.youtube.com/embed/86XQ-Kg5fJ4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<a name="postregionals"></a><br />
<h2><u>Follow-Up Interviews</u></h2><br />
<p>Our second iteration of interviews were conducted once we had a more concrete product built. The purpose of these interviews was to see whether we had successfully addressed the concerns of the first iteration interviews. We also wanted to see whether any new issues with the design existed, which would provide us with potential future directions to take FRED and OSCAR. Kelly Roberge, an independent oil consultant, suggested we look into various ways to deal with the clay and silt particles that can enter our bioreactor system, which can be a major problem since mature fine tailings have a thick consistency that could clog the system.</p><br />
<br />
<h3>Kelly Roberge, of K. Roberge Consulting Ltd. Discussing Bioreactor Improvements</h3><br />
<p>We spoke to Kelly Roberge of K. Roberge Consulting Ltd. who is an independent consultant for the oil sands focusing on mature fine tailings (MFT). He mentioned that in the past 4 years, there has been an increase in looking at biological techniques in the oil sands for remediation, both in understanding natively present microbial life as well as introducing engineered systems.</p><br />
<p> The major concerns that he had with our design at this point were issues with scale-up. These were things such as the amount of toxins that would need to be added to the system to provide constant production of our product, residence time in the bioreactor, as well as the ability for our system to be scaled up to an industrial size. Though we still have much research to do towards this goal of reaching industrial capacity, we did a model scale-up experiment of OSCAR by growing the PetroBrick containing <i>E. coli</i> in our model bioreactor system. The results of this experiment can be seen on the synergy page.</p><br />
<p>In addition, there was a concern raised with the composition of the tailings themselves, due to the mature fine tailings sludge (MFT). In the future we will have to look at the limitations in terms of the capacity of OSCAR to deal with these MFT components. Some suggestions that were made would be to utilize OSCAR in parallel with MFT settling techniques or with runoff water from the tailings drying processes. The sensitivity of our system to this grime and to bitumen would also have to be evaluated and made compatible with the substrates we will be adding in to the system.</p><br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/e5ePaqw5zk4" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>William Sawchuk, of ARC Resources</h3><br />
<p>William Sawchuk, a reservoir engineer at Arc resources, agreed to talk with us about the main parts of our project. This interview confirmed that biological methods, and specifically our project, are definite possibilities of remediation in the oil sands if they can prove to be faster and less harmful than current methods. One concern that William brought up was that there needs to be extra safety factors put in place to avoid posing danger to the environment. This again, serves to further validate the approach that we took to safety, designing both structural and genetic killswitch devices. In the later part of our project, we have also been trying to work on establishing a glycine auxotrophic killswitch to add yet another layer of safety which we feel is necessary. </p><br />
<br />
<p>Another thing Mr. Sawchuk brought up was cost and that it’s important to consider how much a scale-up of the project would cost and if this is less expensive than the current remediation methods. To this end, we’ve been experimenting with starting to get our bioreactor working and have performed an initial validation assay that we can use it in conjunction with our belt skimmer to produce and harvest hydrocarbons. The next step is to scale-up further! The exact cost is a bit tricky. Since the conversion of toxins in the tailings ponds into useful hydrocarbons is a relatively novel idea, it is somewhat difficult to analyze what the cost of a scale-up would be at this point. This is an extremely important future direction for us however.</p><br />
<br />
<br />
<br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/nLeupM1Ype8" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<h3>Gordon Lambert, VP Sustainable Development at Suncor Inc.</h3><br />
<div align="center"><br />
<iframe width="600" height="450" src="http://www.youtube.com/embed/7KbEjQVUsFA" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
</div><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T00:33:09Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magenisum riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. In addition, one of our two 'toxin-sensing' transposon hits was characterized electrochemically, demonstrating its ability to respond to and report on NAs at levels detectable by our electrochemical reporting system. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> and <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> pages respectively. </p></li><br />
<br />
<li><p> Performed an actual test of our biosensor using tailings pond water, showing that we can detect toxins found in tailings. In addition, performed an actual "field test" of our prototype to demonstrate its feasibility and ease of use outside a laboratory setting. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. In addition, we performed an actual validation assay of our bioeacotr, showing that we can use it to grow hydrocarbon producing cells and use our belt skimming device to harvest the hydrocarbons. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<li><p>Performed initial assays on a glycine knockout strain of <i>E. coli</i>, characterizing its survival in differing concentrations of glycine, its ability to work in conjunction with one of our inducible killswitch constructs (<a href=blue href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>) and finally its ability to work with the Petrobrick, actually substantially increasing our out put of hydrocarbons when grown with glycine as compared to a <i>DH5alpha</i> strain. This data can be found on our <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> page. </li></p><br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
<br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T00:21:37Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magenisum riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. In addition, one of our two 'toxin-sensing' transposon hits was characterized electrochemically, demonstrating its ability to respond to and report on NAs at levels detectable by our electrochemical reporting system. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> and <a class="purple" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> pages respectively. </p></li><br />
<br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<li><p>Performed initial assays on a glycine knockout strain of <i>E. coli</i>, characterizing its survival in differing concentrations of glycine, its ability to work in conjunction with one of our inducible killswitch constructs (<a href=blue href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>) and finally its ability to work with the Petrobrick, actually substantially increasing our out put of hydrocarbons when grown with glycine as compared to a <i>DH5alpha</i> strain. This data can be found on our </li></p><br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
<br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T00:13:23Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magenisum riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. In addition, one of our two 'toxin-sensing' transposon hits was characterized electrochemically, demonstrating its ability to respond to and report on NAs at levels detectable by our electrochemical reporting system. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> and <a class="green" href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a> pages respectively. </p></li><br />
<br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T00:08:14Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magenisum riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-27T00:06:56Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magenisum riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p>A β-galactosidase (LacZ) inducible generator construct existing in the registry (<a class="green" href="http://partsregistry.org/Part:BBa_I732901">BBa_I732901</a>) was found to possess a frameshift mutation, affecting its functionality. This part was replaced with a new circuit (<a class="green" href="http://partsregistry.org/Part:BBa_K902090">BBa_K902090</a>), which was characterized for functionality both qualitatively as well as electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.<br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-26T23:50:58Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magenisum riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>). This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-26T23:50:18Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible (<i>Prha</i>) promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>).</li></p><br />
<br />
<li><p>The magenisum riboswitch (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>The <i>Prha</i> promoter was characterized via fluorescence output using a GFP composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902066">BBa_K902066</a>) This promoter was additionally characterized with our S7 kill gene as a composite part (<a href="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p></li></p> <br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli</i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-26T23:40:10Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>). One of these riboswitches (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
<li><p>An <I>E. coli</i> catalase gene from the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_K137068">BBa_K137068</a>) was also tested in conjunction with a lacI inducible promoter as a new composite part (<a class="blue" href= "http://partsregistry.org/Part:BBa_K902060">BBa_K902060</a>) . This part was characterized in TOP10 <i> E. coli<i> for its ability to allow cells to survive in higher concentrations of hydrogen peroxide. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page. <br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/DataPageTeam:Calgary/Project/DataPage2012-10-26T23:29:14Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Detect and Destroy: Data Page|<br />
CONTENT=<br />
<html><br />
<head><br />
<style><br />
<br />
a.green{<br />
color: #159900 !important;<br />
}<br />
a.blue{<br />
color: #1088CC !important;<br />
}<br />
a.orange{<br />
color: #FF7A00 !important;<br />
}<br />
<br />
</style><br />
</head><br />
<br />
<body><br />
<div align="justify"><br />
<br />
<br />
<a name="top"></a><br />
<br />
</html>[[File:UCalgary2012_GraphicalAbstract.png|thumb|700px|center|thumb|Figure 1. The Calgary team has developed a dual system for the detection of toxic components in tailing ponds and the remediation of these compounds. Tailings ponds are large bodies of water containing waste products produced from the extraction of bitumen in the oil sands. Our biosensor involved the identification of a toxin promoter through a transposon screen. An electrochemical detector was implemented with a multiple output system allowing for the detection of multiple compounds simultaneously. This promoter/detector system was then complemented with the production of a biosensor prototype involving both a physical device and a software program for easy data analysis.<br />
<br />
Rather than just sensing toxins in the tailings ponds, a major objective was to detoxify the tailings through the reduction of toxins such as carboxylic acids (mainly naphthenic acids) and catechol, turning them into usable hydrocarbons. Purification of these hydrocarbons would contribute an added economic and industrial benefit. In order to house this system, we also aimed to design a bioreactor for our bacteria as well as optimize product output through a flux-variability based model. Finally, in order to create higher quality hydrocarbons, we explored desulfurization and denitrogenation pathways to upgrade our fuel. To do this in a safe and environmentally sound manner, we built into our design structural containment, as well as genetic control mechanisms through novel inducible ribo-killswitches.]]<html> <br />
<br />
<a name="newparts"></a><br />
<h2>Characterization of new parts submitted to the Registry</h2><br />
<br />
<ul><li><p>(<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902000">BBa_K902000</a>) and (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902004">BBa_K902004</a>): two novel hydrolase enzymes were submitted to the registry for the hydrolysis of two different sugar-conjugated electroactive compounds: PNPG and PDPG. Used in conjunction with the existing lacZ part in the registry (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I732005">BBa_I732005</a>) which hydrolyzes CPRG, this allows for the electrochemical detection of three compounds with a single electrode. A <i>uidA</i> inducible generator (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902002">BBa_K902002</a>) was submitted and characterized electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<br />
<li><p>(<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>),(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902023">BBa_K902023</a>) and (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902074">BBa_K902074</a>): three novel riboswitches sensitive to magnesium, molybdate, and manganese were submitted along with two associated promoters (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902009">BBa_K902009</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902073">BBa_K902073</a>) in addition to a rhamnose inducible, glucose repressible promoter (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">BBa_K902065</a>). One of these riboswitches (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902008">BBa_K902008</a>) was tested with GFP and a constitutive promoter using this construct, (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902021">BBa_K902021</a>), with its promoter and GFP using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902017">BBa_K902017</a>) and with its promoter and the <i>S7</i> kill gene using this construct (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902018">BBa_K902018</a>). This data can be found on our killswitch <a class="orange" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a> page. </li></p><br />
<br />
<li><p>Genes for denitrogenation and desulfurization were biobricked and submitted. The <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation"><i>amdA</i></a>, amidase gene <a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902041">(BBa_K902041)</a> was biobricked and characterized shown to be able to remove primary amines from a variety of compounds. A novel oxidoreductase part (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902058">BBa_K902058</a>) was also submitted and its functionality characterized for use in the desulfurization project. This data can be found on our upgrading <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a> page.</p></li></ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<a name="existingparts"></a><br />
<h2>Further characterization of parts already present within the registry </h2><br />
<br />
<ul><li><p>The IPTG inducible lacI regulated promoter (<a class="green" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010">BBa_R0010</a>) was tested electrochemically to demonstrate its leakiness when not used in conjunction with strong expression of regulatory elements. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</li><br />
<li><p>(<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>), the PetroBrick, submitted by the Washington team in 2011, was characterized for a novel function: the conversion of naphthenic acids and 2-hydroxymuconate- a catechol break-down product from from the xylE gene (<a class="blue" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) into hydrocarbons and potential value added products. This data can be found on both the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> page and the <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. We feel that these new and meaningful applications of this part present a distinct improvement on its usefulness for other teams.</li><br />
<br />
<li><p> The output of (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) was also optimized thorugh a program we developed in MATLAB for the optimization of metabolic pathways in synthetic biology metabolic networks. The program allows you to build an artificial synthetic biology network in <i>E. coli</i> and predicts substrates that should be fed to the organism to increase production of the compound. This was characterized and validated in the wetlab with the Petrobrick. This data can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a> page.</li><br />
<br />
<li><p> An existing <i>xylE</i> gene in the registry (<a class="blue" href= "http://partsregistry.org/Part:BBa_J33204">BBa_J33204</a>) was constructed with a constitutive promoter instead of the glucose-repressible part available within the registry. This allows for increased output in media containing glucose, making it more suitable for a variety of applications such as our own. We validated the functionality of this part which can be found on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a> page. In addition, we documented a novel application for this part, by using it in conjunction with Washington's PetroBrick (<a class="blue" href="http://partsregistry.org/Part:BBa_K590025">BBa_K590025</a>) to degrade catechol into a further break-down product. </p></li><br />
<br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
<br />
<br />
<a name="additionalwork"></a><br />
<h2>Additional Work and Characterization </h2><br />
<br />
<ul><br />
<li><p>Developed and tested both hardware and software for a biosensor using an electrochemical sensor. The software is available on our wiki as are the results fom the hardware. These are on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a> page</p></li><br />
<br />
<li><p> Characterized one of our constitutively expressed transposon clones to test the lacZ gene electrochemically. This data can be found on our <a class="green" href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a> page.</p></li><br />
<li><p> Submitted novel parts involved in decarboxylation and validated the functionality of an additional enzyme (oleT), capable of converting fatty acids into alkenes by itself. This was done in its host organism. This data can be found in our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a> section, however this gene has not yet been submitted due to problems cloning it.</p></li><br />
<br />
<li><p>Designed and prototyped a physical bioreactor for which we obtained both qualitative and quantitative data for its functionality. This is outlined on our <a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a> page. </p></li><br />
<br />
<li><p>Characterized the biodegradation of carbazole and various sulfur-containing compounds resembling naphthenic acids in the organisms from which we got our genes. This data can be found on our (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a>) and (<a class="blue" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a>)</p></li><br />
<br />
<br />
<br />
<br />
<li><p>Resubmitted (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K206009">BBa_K26009</a>) an inconsistent registry composite part that we had to construct from basic parts, resubmitting as the sequence-verified (<a class="orange" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902016">BBa_K902016</a>)</p></li><br />
</ul><br />
<p><a href="#top">Back to Top</a></p><br />
</body><br />
<br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2012-10-26T09:24:04Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectOrange|<br />
TITLE=Project Overview|CONTENT=<br />
<html><br />
<head><br />
<style><br />
#fredbox{<br />
width: 320px;<br />
height: 215px;<br />
background: #58CD45;<br />
margin-left: 30px;<br />
float:left;<br />
}<br />
#fredbox:hover{<br />
background: #94FF7D;<br />
}<br />
#oscarbox{<br />
width: 320px;<br />
height: 215px;<br />
background: #5BB5E8;<br />
margin-left: 4px;<br />
float:left;<br />
}<br />
#oscarbox:hover{<br />
background: #7DD7FF;<br />
}<br />
</style><br />
</head><br />
<body><br />
<h2>Toxins In Our Environment</h2><br />
<p>During petroleum extraction and refinement processes, toxic byproducts are produced. These compounds have created enormous environmental disturbances, burdening our ecosystems with land, water, and air contamination. <br />
Common forms of air pollutants consist of NO<sub>x</sub> (nitrogen containing compounds) and SO<sub>x</sub> (sulfur containing compounds) which contribute to green house gas accumulation and acid rain (Schneider, 2006; Environmental protection agency, 1999). <br />
<br />
Similarly, land and water contaminants often consist of complex mixtures including highly toxic phenols and aromatic compounds, toxic and corrosive carboxylic acids (naphthenic acids) as well as sulfur and nitrogen-containing compounds. These often are recalcitrant, having complex structures that are difficult to break down, causing them to persist in the ecosystem. Classical examples of water contamination include tailings ponds, which contain byproducts from the bitumen extraction process of oil sands. Although the water in tailings ponds is recycled to the extraction process, it is not treated to remove the toxins but are merely contained as much as possible. This creates a susceptibility towards contamination of surrounding areas as a result of these toxic compounds leaching into ground water sources, through spills or through the accidental release of waste products into the environment. </p><br />
<br />
<br />
<br />
</html>[[Image:Calgary_EnviroToxins.jpg|thumb|600px|center|Figure 1: Environmental toxins contaminate air, water, and land masses. These can consist of various compounds which could be divided into sulfur, nitrogen, carboxylic acid, and phenolic based compounds. What can we do to solve this problem?]]<html><br />
<br />
<h2>Synthetic Biology As A Platform For Remediation</h2><br />
<br />
<p>The removal of these compounds is becoming a more and more pressing issue, especially as government bodies start to become more proactive, implementing stricter regulation. Presently, there are a variety of solutions to remove these compounds from the environment using chemical means. These methods involve the use of chemical agents or the physical removal of contaminated soil or water samples and storing these products in contained areas (Scott <i>et al</i>. 2005). There is still however, no efficient, environmentally friendly mechanism for this to occur. The real question is,</p><br />
<br />
<p><b>What do we need in order to remediate these toxins from the environment?</b></p><br />
<br />
<p>We require a method to be able to easily and economically detect where these toxins are and then look to remediating them. Interestingly, microorganisms in the environment have evolved to be able to do both of these functions, responding to compounds in their environment and transforming them into food or other products. Harnessing these natural mechanisms through an engineered synthetic biology could thus be a viable option.</p><br />
<br />
<p><b>What if we could detect toxins in our environment using a synthetically engineered organism? What if we could use a second organism to take these compounds and not only <u>degrade</u> them but convert them into <u>useful</u> compounds like hydrocarbons!</b></p><br />
<br />
<h2>Introducing...</h2><br />
<br />
<br />
</html>[[File:Calgary FredandOscarDef.jpg|thumb|600px|center|Figure 2: Introducing our dynamic duo FRED and OSCAR! This biosensor/bioreactor team is ready to detect and remediate toxins in the environment. Not only can OSCAR break down toxic carboxylic acid containing compounds such as naphthenic acids, but we also demonstrated that he can turn them into functional hydrocarbons!]]<html><br />
<br />
<p><br />
We would like to introduce FRED and OSCAR! Our dynamic biosensor/bioreactor duo designed to be able to detect toxic compounds such as the ones illustrated above in liquid waste and contaminated waters and also be able to convert these toxic components into useable hydrocarbons. FRED, the Functional Robust Electrochemical Detector, is capable of detecting various toxic components simultaneously through an electrochemical response. Building on the single output <a class="blue" href="https://2011.igem.org/Team:Calgary">biosensor for NAs</a> that we developed last year, we set out to design a multiple output biosensor. We illustrated how this sensor could work by showing that it has the potential to detect multiple toxins in contaminated water. Additionally, we developed a miniaturized circuit for a prototype, validated that this device worked in the wetlab, and designed our own software available to everyone to be used with a home made potentiostat. <br />
</p><br />
<p><br />
OSCAR, the Optimized System for Carboxylic Acid Remediation, is designed specifically to target toxins such as naphthenic acids (carboxylic acid-containing compounds), catechol, and nitrogen and sulfur containing heterocycles. Using the PetroBrick (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025">BBa_K590025</a>) we were able to convert various naphthenic acid based compounds into their hydrocarbon analogs. Additionally, we wanted to be able to degrade other toxic components of tailings so we used the <i>xylE</i> gene (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J33204">BBa_J33204</a>) in order to cleave catechol, an abundant intermediate in many toxic areas. Not only did we set out to break down catechol, but we attempted to see if we could further reduce the toxicity of the catechol breakdown product through use of the PetroBrick. When we co-culture these genetic circuits we can selectively produce new compounds from catechol compared to with <i>xylE</i> alone, suggesting that the Petrobrick may be used to create new hydrocarbon based compounds! Lastly we wanted to remove sulfur and nitrogen from heterocycles using the <i>dsz</i> and <i>carA</i> operons respectively. Not only would this improve the quality of fuel produced, but also prevent the production of NO<sub>x</sub> and SO<sub>x</sub> during combustion, reducing the amount of air pollution produced from burning fuel. </p><br />
<br />
<h2>Taking A Step Back - Human Practices Inspired Our Project!</h2><br />
<img src="https://static.igem.org/mediawiki/2012/1/17/UCalgary2012_FRED_and_OSCAR_HP.png" style="float: right; width: 200px; padding: 10px;"></img><br />
<p>Before starting our project, the Calgary iGEM team felt it would be important to <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">answer a few questions</a> about how FRED and OSCAR could be applied into the oil and gas sector.</p> <br />
<br />
<p><b>Would oilsands industry be interested in a biosensor and bioreactor for remediation purposes?</b> Yes! In fact, our meeting with the Oilsands Leadership Initiative (OSLI) has led us to believe that industry is interested in potentially using synthetic biology for remediation of toxins.</p> <br />
<p><b>What would people think about using synthetic biology<img src="https://static.igem.org/mediawiki/2012/e/e8/UCalgary2012_FRED_and_OSCAR_Interviews_Low-Res.png" style="float: right; padding: 10px; width: 200px;"></img> in the oilsands? Do they have any concerns about its implementation?</b> We consulted with two professionals working in biotechnology and ecological development in Alberta. Both of them made it clear that while the concept sounds great its important that we keep in mind the safety and ethics of our project.</p> <br />
<br />
<p><b>How can OSCAR and FRED be designed with safety in mind?</b> From our various conversations our team looked towards both physical <img src="https://static.igem.org/mediawiki/2012/c/c3/UCalgary2012_FRED_and_OSCAR_Design.png" style="float: right; padding: 10px; width: 200px;"></img>and genetic design considerations to ensure that both FRED and OSCAR were designed form the beginning in a safe and functional way. This involved developing biosensor and bioreactor containment devices as well as kill switch.</p> <br />
<br />
<p><b>How can we teach people more about FRED, OSCAR, and Synthetic Biology?</b> From our interviews it was clear that not many people knew much about synthetic biology or its applications in the oil and gas sector. For this we partnered with the Telus Spark Centre, the local Science Centre in Calgary to help communicate synthetic biology to them. We also developed a video game that we took to the centre and better educated adults and kids on synthetic biology! </p><br />
<br />
<h2>Learn More About FRED and OSCAR</h2><br />
<p>To learn more about our team see the <a href="https://2012.igem.org/Team:Calgary/Project/DataPage">data page</a>, or the <a href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a> and <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a> overview pages below.</p><br />
<br />
<a href="https://2012.igem.org/Team:Calgary/Project/FRED"><div class="imgbox" id="fredbox"><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/UCalgary2012_EpicBoxFRED_-_Blank.png"></img><br />
</div></a><br />
<a href="https://2012.igem.org/Team:Calgary/Project/OSCAR"><div class="imgbox" id="oscarbox"><br />
<img src="https://static.igem.org/mediawiki/2012/9/94/UCalgary2012_EpicBoxOSCAR_-_Blank.png"></img><br />
</div></a><br />
</body><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T09:19:21Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<br><br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Electrochemically characterized</b> an inducible <b><i>lacz</i> circuit</b>, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><b>Electrochemically characterized</b> a constitutive <b>bglX generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><b>Characterized</b> and demonstrated how <b>3 different hydrolase-producing circuits</b> can be used simultaneously in order to create a <b>multiplex biosensor.</b> </p></li><br />
<br />
<li><p><b>Tested one of our transposon library hits</b> with our electrochemical reporter, showing that we can use our system to <b>detect toxins electrochemically</b>.</p></li><br />
</ul><br />
<br />
<br><br />
<br />
<p><b>In terms of <FONT COLOR=#1088CC>OCSAR</FONT>, we...</b></p><br />
<ul><br />
<li><p>Obtained <b>additional characterization data</b> in validation of our flux-variability analysis model.</p></li><br />
<br />
<li><p>Characterized the ability of our <b>novel amidase BioBrick</b> to remove nitrogen atoms from ring structures with remarkeable efficiency, turning them into a substrate that the Petrobrick (BBa_K) can likely convert into hydrocarbons.</p></li><br />
<br />
<li><p>Tested out our bioreactor and it’s ability to grow cells containing the PetroBrick as well as extract the hydrocarbon layer produced using our belt skimmer</p></li><br />
</ul><br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T09:14:48Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<br><br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Characterized</b> an inducible <b><i>lacz</i> circuit</b>, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><b>Characterized</b> a constitutive <b>bglX generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><b>Characterized</b> and demonstrated how <b>3 different hydrolase-producing circuits</b> can be used simultaneously in order to create a <b>multiplex biosensor.</b> </p></li><br />
<br />
<li><p><b>Tested one of our transposon library hits</b> with our electrrchemical reporter, showing that we can use our system to detect toxins electrochemically</p></li><br />
</ul><br />
<br />
<br><br />
<br />
<p><b>In terms of <FONT COLOR=#1088CC>OCSAR</FONT>, we...</b></p><br />
<br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T09:13:28Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<br><br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Characterized</b> an inducible <b><i>lacz</i> circuit</b>, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><b>Characterized</b> a constitutive <b>bglX generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><b>Characterized</b> and demonstrated how <b>3 different hydrolase-producing circuits</b> can be used simultaneously in order to create a <b>multiple biosensor.</b> </p></li><br />
<br />
<li><p><b>Tested one of our transposon library hits</b> with our electrrchemical reporter, showing that we can use our system to detect toxins electrochemically</p></li><br />
</ul><br />
<br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T09:13:02Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<br><br><br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Characterized</b> an inducible <b><i>lacz</i> circuit</b>, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><b>Characterized</b> a constitutive <b>bglX generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><b>Characterized</b> and demonstrated how <b>3 different hydrolase-producing circuits</b> can be used simultaneously in order to create a <b>multiple biosensor.</b> </p></li><br />
<br />
<li><p><b>Tested one of our transposon library hits</b> with our electrrchemical reporter, showing that we can use our system to detect toxins electrochemically</p></li><br />
</ul><br />
<br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T09:12:30Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Characterized</b> an inducible <b><i>lacz</i> circuit</b>, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><b>Characterized</b> a constitutive <b>bglX generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><b>Characterized</b> and demonstrated how <b>3 different hydrolase-producing circuits</b> can be used simultaneously in order to create a <b>multiple biosensor.</b> </p></li><br />
<br />
<li><p><b>Tested one of our transposon library hits</b> with our electrrchemical reporter, showing that we can use our system to detect toxins electrochemically</p></li><br />
</ul><br />
<br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T09:11:45Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<br />
<li><p><b>Characterized</b> an inducible <b><i>lacz</i> circuit</b>, replacing a faulty existing circuit in the registry containing a frameshift.</p></li><br />
<br />
<li><p><b>Characterized</b> a constitutive <b>bglX generator</b> in terms of its electrochemical activity.</p></li><br />
<br />
<li><p><b>Characterized</b> and demonstrated how <b>3 different hydrolase-producing circuits</b> can be used simultaneously in order to create a <b>multiple biosensor.</b> </p></li><br />
<br />
<li><p><b>Tested one of our transposon library hits</b> with our electrrchemical reporter, showing that we can use our system to detect toxins electrochemically</p></li><br />
<br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T09:01:54Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<p><b>In terms of <FONT COLOR=#159900>FRED</FONT>, we...</b></p><br />
<br />
<h2> <br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:48:04Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium riboswitch kill gene construct</b>.</li></p></ul><br />
<br />
<p><b>In terms of <FONT COLOR="FF7A00">FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:42:09Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<li><p><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<li><p><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<li><p><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li></p></ul><br />
<br />
<p><b>In terms of <FONT COLOR="FF7A00">FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:41:41Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<p><li><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li><p>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<p><li><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li></p></ul><br />
<br />
<p><b>In terms of <FONT COLOR="FF7A00">FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:41:10Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<p><li><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<p><li>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<p><li><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li></p></ul><br />
<br />
<p><b>In terms of <FONT COLOR="FF7A00">FRED</FONT>, we...</b></p><br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:40:39Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<p><li><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<p><li>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<p><li><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li></p></ul><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:39:39Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<p><li><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<p><li>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li></p><br />
<br />
<p><li><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li></p></ul><br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:38:53Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li></p><br />
<br />
<p><li><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li></p><br />
<br />
<br />
<li>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li><br />
<br />
<li><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li></ul><br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:38:14Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<ul><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li><br />
<br />
<li><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li><br />
<br />
<br />
<li>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li><br />
<br />
<li><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li></ul><br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:37:29Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li><br />
<br />
<li><b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.</li><br />
<br />
<br />
<li>Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene.</li><br />
<br />
<li><b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.</li><br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:36:36Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<br />
<p><li><b>Returned to industry experts</b> to see if we accomplished our goals and what the <b>next steps</b> should be.</li><br />
<br />
<b>Characterized</b> the functionality of our previously submitted <b><i>Prha</i> promoter</b> using GFP fluorescence.<br />
<br />
<br />
Tested an additional <b>complete kill switch device</b> using the <i>Prha</i> promoter with our S7 kill gene. <br />
<br />
<b>Further characterized</b> our previously validated <b>magnesium rioboswitch construct</b>.<br />
</p><br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/OSCARTeam:Calgary/Project/OSCAR2012-10-26T08:28:13Z<p>Emily Hicks: </p>
<hr />
<div>{{Team:Calgary/TemplateProjectBlue|<br />
TITLE=OSCAR|<br />
<br />
CONTENT=<html><br />
<head><br />
<style> <br />
#hub_oscar1{<br />
background: #5BB5E8;<br />
}<br />
#hub_oscar2{<br />
background: #50A2D0;<br />
}<br />
#hub_oscar3{<br />
background: #4292BF;<br />
}<br />
#hub_oscar4{<br />
background: #3285B4;<br />
}<br />
#hub_oscar5{<br />
background: #2580B3;<br />
}<br />
#hub_oscar1:hover, #hub_oscar2:hover, #hub_oscar3:hover, #hub_oscar4:hover, #hub_oscar5:hover{<br />
background: #7DD7FF;<br />
}<br />
<br />
</style><br />
</head><br />
<body><br />
</html>[[IMAGE:UCalgary2012_OSCAR+definition.png|268px|right]]<html><br />
<br />
<p>The <b>O</b>ptimized <b>S</b>ystem for <b>C</b>arboxylic <br />
<br />
<b>A</b>cid <b>R</b>emediation, or OSCAR, is the <br />
<br />
<i><b>Destroy</i></b> component to our iGEM 2012 Calgary project. <br />
<br />
With our detection system in place, OSCAR converts toxic compounds, such as <br />
<br />
naphthenic acids and catechol, into hydrocarbons by removing <br />
<br />
unwanted carboxylic acid and hydroxyl groups. <br />
<br />
</p><br />
<br />
<p> By conversion to hydrocarbons we can not only detoxify tailing <br />
<br />
waters but provide an economically viable method for doing so. By <br />
<br />
using flux balance analysis we developed a system to optimize the <br />
<br />
output of carboxylic acid removal system which we validated in the <br />
<br />
wetlab. Furthermore we developed a bioreactor prototype to <br />
<br />
demonstrate the applicability of our system using novel <br />
<br />
hydrocarbon collection methodologies. Finally, we developed <br />
<br />
constructs and genetic circuits to upgrade these hydrocarbons to <br />
<br />
reduce sulfur and nitrogen content. Altogether, OSCAR provides a <br />
<br />
method to upgrade naphthenic acids and other toxic components from <br />
<br />
waste products into useable fuels.</p><br />
<br />
<h2>What is OSCAR composed of?</h2><br />
<br />
<br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation"><br />
<div class="hubbox" id="hub_oscar1"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/UCalgary2012_IconOSCAR1.png"></img><br />
<h2>Decarboxylation</h2><br />
<p>The major goal of OSCAR was to be able to remediate toxic components in oil sand tailings ponds, tuning them into useable hydrocarbons. We targeted </html>'''carboxylic acid remediation'''<html>, such as naphthenic acids, as they are some of the most toxic components of the ponds. We had a couple approaches to this, with some promising results!</p><br />
</div><br />
</a><br />
<br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation"><br />
<div class="hubbox" id="hub_oscar2"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/UCalgary2012_IconOSCAR4.png"></img><br />
<h2>Catechol Degradation</h2><br />
<p>Another toxin we wanted to target was catechol, as it is the common breakdown product of many different toxic compounds. Through modifications to existing registry parts, we showed that we could </html>'''degrade catechol'''<html> into 2HMS and then further degrade it into hydrocarbons.</p><br />
</div><br />
</a><br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><br />
<div class="hubbox" id="hub_oscar3"><br />
<img src="https://static.igem.org/mediawiki/2012/4/40/UCalgary2012_IconOSCAR6.png"></img><br />
<h2>Flux Analysis</h2><br />
<p>Once we had our bacteria producing hydrocarbons out of tailings pond toxins, we wanted to figure out how to optimize their efficiency. We developed a program to optimize the metabolic network of our synthetic organism using a </html>'''mathematical model'''<html> which predicts compounds that could be fed to the organism to increase hydrocarbon production. Once complete, we validated it in the wetlab, and developed a graphical user interface using the matlab platform, allowing all iGEM teams to use it in their application.</p><br />
</div><br />
</a><br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor"><br />
<div class="hubbox" id="hub_oscar4"><br />
<img src="https://static.igem.org/mediawiki/2012/3/3e/UCalgary2012_IconOSCAR5.png"></img><br />
<h2>Bioreactor</h2><br />
<p>With our biological systems showing some promising results in terms of decarboxylation, we needed to think about where OSCAR could live. He needed a house: a </html> '''bioreactor!''' <html>We first used Maya to create a model animation, and then designed and prototyped it. We tested its functionality in terms of a few different parameters, trying to find the most efficient design. </p><br />
</div><br />
</a><br />
<br />
<a class="hublink" href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading"><br />
<div class="hubbox" id="hub_oscar5"><br />
<img src="https://static.igem.org/mediawiki/2012/4/44/UCalgary2012_IconOSCAR7.png"></img><br />
<h2>Oil Upgrading</h2><br />
<p>Once we knew that we could turn carboxylic acids into hydrocarbons, we wanted to explore how to upgrade them. Sulfur and nitrogen are both compounds abundant in the tailings ponds and that reduce the quality of fuels produced. They also produce toxic compounds when burned, making them undesirable. We explored ways to remove these components through </html> '''desulfurization''' and '''denitrogenation'''<html>. We targeted pathways in a variety of organisms and submitted biobricks for each.</p><br />
</div><br />
</a><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</body><br />
</html><br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-26T08:16:12Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<br />
<h2>Incorporating human practices in the design of our system </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. 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 wok 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 />
<br />
<br />
<br />
<br />
<br />
</html><br />
<br />
<br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:15:12Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team has had many accomplishments since the regional jamboree!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<br />
</html><br />
}}</div>Emily Hickshttp://2012.igem.org/Team:Calgary/Project/Post-RegionalsTeam:Calgary/Project/Post-Regionals2012-10-26T08:14:38Z<p>Emily Hicks: </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 />
SECTION = Project|<br />
SIDELIST =<br />
<html><br />
<head><br />
<style><br />
/*colouring: current page and all sidebar rollovers*/<br />
#projectlink, #sidebar #list a:hover, #nav li a:hover, #nav li a.drop:hover::after{<br />
color: #DF43FF !important;<br />
}<br />
/*colouring: sidebar border*/<br />
#sidebar #list{<br />
border-left: 10px solid #C6D !important;<br />
}<br />
/*colouring: links*/<br />
#bodycontainer a{<br />
color: #C5B !important;<br />
}<br />
#bodycontainer a:visited{<br />
color: #C4A;<br />
}<br />
</style><br />
</head><br />
<br />
<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 />
<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 />
<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=Post-Regional Accomplishments|CONTENT=<br />
<html><br />
<br />
<h2> Our team had many accomplishments throughout the summer of 2012!</h2><br />
<br />
<p><b>In our <FONT COLOR="FF7A00">Human Practices</FONT> project, we...</b></p><br />
<br />
</html><br />
}}</div>Emily Hicks