http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Rpgguardian&year=&month=2012.igem.org - User contributions [en]2024-03-29T15:48:10ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Calgary/Notebook/ProtocolsTeam:Calgary/Notebook/Protocols2012-10-27T03:54:53Z<p>Rpgguardian: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Protocols|<br />
CONTENT = <html><br />
<p>Here is a list of all the procedures we used this summer. Each contains a description and list of materials required.</p><br />
<ul><br />
<br />
<h2>General Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/agarosegel">Agarose Gel Electrophresis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gemomicprep">Bacterial Genomic DNA Purification</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/transformation">Bacterial Transformation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">Construction Techniques</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole GC-MS Analysis">GC-MS Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gelextraction">Gel Extraction</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson Assembly">Gibson Assembly</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/lbagar">LB Agar Plates</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 Minimal Media Preparation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction">Organic Extraction</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Overnight Cultures</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/pcrpurification">PCR Purification</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/picogreen">PicoGreen Assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">Plasmid Purification (from <i>E. coli</i>)</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/compcells">Preparing Chemically Competent Cells (<i>E. coli</i>)</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">Preparing Glycerol Stocks (<i>E. coli</i>)<br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dnarehydration">Rehydration of Registry DNA</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry">Reviving Freeze-dried Bacterial Cultures from DSMZ</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">Site-Directed Mutagenesis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">Splice Overlap Extension PCR (SOE PCR)</a></li><br />
<br />
<br />
<br />
<h2>Electrochemistry Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">Cyclic Voltammetry</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">Potentiostatic Standard Curve Generation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potentiostatic">Reporter Expression Detection</a></li><br />
<br />
<br />
<h2>Desulfurization Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/catalase">Catalase assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/desulfur">Desulfurization Assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hpac">HpaC assay</a></li><br />
<br />
<h2>Decarboxylation Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/PetroBrick Validation Assay">PetroBrick Validation Assay </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT in Validation Assay">oleT Validation Assay </a></li><br />
<br />
<h2>Denitrogenation Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/AmidaseAssay"><i>AmdA</i> Characterization Assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole">Carbazole Degradation Assay</a></li><br />
<br />
<br />
<h2>Decatecholization Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization">Decatecholization Assay</a></li><br />
<h2>Transposon Mutant Library for Toxin Detection</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">Transposon-Mediated Mutant Library Generation</a></li><br />
<br />
<h2>Kill Switch Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">Characterization of mgtA regulation with GFP </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">Characterization of mgtA regulation with S7 killgene </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Prha Characterization">Characterization of <i>P<sub>rha</sub></i> with GFP</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/escapersassay">Kill Assay for rhamnose system</a></li><br />
</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/GlycineAssays">Glycine Auxotrophic Assays (Glycine Media Test, and Petrobrick Test)<br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/nucleaseassay">Nuclease assay</a></li><br />
</a></li><br />
<br />
<h2>Bioreactor Assays Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/bacgrowth">Bacterial Growth Experiments</a></li><br />
<p>Belt Selection Tests:</p><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/bactest">Bacteria Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hcseptest">Hydrocarbon Separation Test</a></li><li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hctest">Hydrocarbon Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hdbeltskim">NA and Hexadecane Belt Skim Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tailingtest">Tailings Test</a></li><br />
</ul><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<h2>Modeling Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Modelvalidation">Modelling validation experiments</a></li><br />
<br />
<br />
<br />
</ul><br />
<br />
</html><br />
}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Notebook/ProtocolsTeam:Calgary/Notebook/Protocols2012-10-27T03:54:16Z<p>Rpgguardian: </p>
<hr />
<div>{{Team:Calgary/TemplateNotebookOrange|<br />
<br />
TITLE=Protocols|<br />
CONTENT = <html><br />
<p>Here is a list of all the procedures we used this summer. Each contains a description and list of materials required.</p><br />
<ul><br />
<br />
<h2>General Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/agarosegel">Agarose Gel Electrophresis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gemomicprep">Bacterial Genomic DNA Purification</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/transformation">Bacterial Transformation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/construction">Construction Techniques</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole GC-MS Analysis">GC-MS Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/gelextraction">Gel Extraction</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Gibson Assembly">Gibson Assembly</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/lbagar">LB Agar Plates</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 Minimal Media Preparation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oextraction">Organic Extraction</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Overnight Cultures</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/pcrpurification">PCR Purification</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/picogreen">PicoGreen Assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/plasmidminiprep">Plasmid Purification (from <i>E. coli</i>)</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/compcells">Preparing Chemically Competent Cells (<i>E. coli</i>)</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/glycerolstock">Preparing Glycerol Stocks (<i>E. coli</i>)<br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dnarehydration">Rehydration of Registry DNA</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/dmszfreezedry">Reviving Freeze-dried Bacterial Cultures from DSMZ</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis">Site-Directed Mutagenesis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/soe">Splice Overlap Extension PCR (SOE PCR)</a></li><br />
<br />
<br />
<br />
<h2>Electrochemistry Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/cvs">Cyclic Voltammetry</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potstd">Potentiostatic Standard Curve Generation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/potentiostatic">Reporter Expression Detection</a></li><br />
<br />
<br />
<h2>Desulfurization Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/catalase">Catalase assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/desulfur">Desulfurization Assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hpac">HpaC assay</a></li><br />
<br />
<h2>Decarboxylation Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/PetroBrick Validation Assay">PetroBrick Validation Assay </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT in Validation Assay">oleT Validation Assay </a></li><br />
<br />
<h2>Denitrogenation Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/AmidaseAssay"><i>AmdA</i> Characterization Assay</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/carbazole">Carbazole Degradation Assay</a></li><br />
<br />
<br />
<h2>Decatecholization Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization">Decatecholization Assay</a></li><br />
<h2>Transposon Mutant Library for Toxin Detection</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">Transposon-Mediated Mutant Library Generation</a></li><br />
<br />
<h2>Kill Switch Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">Characterization of mgtA regulation with GFP </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">Characterization of mgtA regulation with S7 killgene </a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Prha Characterization">Characterization of <i>P<sub>rha</sub></i> with GFP</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/escapersassay">Kill Assay for rhamnose system</a></li><br />
</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/GlycineAssays">Glycine Auxotrophic Assays (Glycine Media Test, Petrobrick Test, and Killswitch Testing)<br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/nucleaseassay">Nuclease assay</a></li><br />
</a></li><br />
<br />
<h2>Bioreactor Assays Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/bacgrowth">Bacterial Growth Experiments</a></li><br />
<p>Belt Selection Tests:</p><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/bactest">Bacteria Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hcseptest">Hydrocarbon Separation Test</a></li><li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hctest">Hydrocarbon Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/hdbeltskim">NA and Hexadecane Belt Skim Test</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/tailingtest">Tailings Test</a></li><br />
</ul><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<h2>Modeling Protocols</h2><br />
<li><a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Modelvalidation">Modelling validation experiments</a></li><br />
<br />
<br />
<br />
</ul><br />
<br />
</html><br />
}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:34:52Z<p>Rpgguardian: </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 />
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</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 />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED to show that the prototype that we built is feasible and easy to use, we investigated whether it would be permissable or advisable to try FRED outside of the lab. We performed a literature search to look for any regulations that might exist. Nothing pertaining to our province could be found, so we looked to Ontario and the United States. The concise guide to U.S. federal guidelines, rules and regulations for synthetic biology outlined the rules pertaining to field tests and indicated that in cases where organisms are going to be released into the environment, the EPA (environmental protection agency) requires a TSCA (Toxic Substances Control Act) Experimental Release Application (TERA) to be completed 60 days before the trial begins and the APHIS (Animal and Plant Health Inspection Service) requires a permit or notification. Although we specifically designed FRED to not release the microbes but rather to contain them, the prototype is too much in its infancy to remove it from the lab and be <b>absolutely</b> assured that it won’t be released. What we did instead, was took our prototype without bacteria in it to collect a water sample in a nearby river in Calgary. The video of this experience can be found below. </p><br />
<br />
<div align="center"><br />
<iframe width="640" height="360" src="http://www.youtube.com/embed/AFO8sQB1PmE" frameborder="0" allowfullscreen></iframe><br />
</div><br />
<br />
<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 />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:32:01Z<p>Rpgguardian: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
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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 />
<p>This result was extremely exciting for us, as we see clear induction of the system in the presence of tailings, as compared to the control. Although we don't know exactly what we are sensing, (remember that our transposon is sensitive to 3 different toxins: DBT, Carbazole and NAs),we are definitely sensing something! <b>This shows that FRED is functional and more than that, FRED is functional in the application for which he was designed!</b> The next step will be to quantify toxins present in tailings pond water samples in order to calibrate our reporter. </p><br />
<br />
<h2> Taking FRED out to the field! </h2><br />
<br />
<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED 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>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:25:01Z<p>Rpgguardian: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’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. ]]<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>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:08:07Z<p>Rpgguardian: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
{{Team:Calgary/BasicPage|proj_hp|<br />
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<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 />
<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 />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:06:49Z<p>Rpgguardian: </p>
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</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 />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
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<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 />
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.<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 />
<h2> <u>Putting FRED together</u> </h2><br />
<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>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 />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T03:00:27Z<p>Rpgguardian: </p>
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
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<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
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<h2>Have we accomplished our goal?</h2><br />
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<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’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 />
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<p>Here we demonstrate that we can develop a <b>comprehensive kill switch</b> consisting of both an auxotroph and an inducible kill switch which work together to contain FRED and OSCAR. With FRED, we show that we can detect <b>toxins selectively in tailing ponds</b> using our 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 />
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<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
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</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 1: Glycine requirements for growth of <i>glyA</i> knockout strain JW2535-1. The bacteria was grown in LB overnight, washed, and subcultured into M9 minimal media, glucose, with various different concentration of glycine (from 1nM logarithmically to 100 mM). Interestingly, the glycine knockout grew best at concentrations of 1 - 10 mM. However, the auxotroph was not strong enough even at low concentrations to completely abolish growth.]]<html><br />
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<p>As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
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<h2>Testing the Auxotrophic Marker as a Kill Switch</h2><br />
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</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 />
<h2> <u>Putting FRED together</u> </h2><br />
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<h2>Can we sense toxins?</h2><br />
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<p>Now that we’ve been able to show that we can indeed sense three compounds electrochemically and simultaneously using our hydrolase system, and characterized genetic circuits for two of these outputs, our next goal was to actually try to sense toxins. Despite the fact that we have encountered significant difficulty in trying to sequence our transposon clones, given that we designed our transposon library to use <i>lacZ</i>, we could actually use our transposon directly in our electrochemical reporter system without actually knowing the identity of the sensory element. Although we do plan to BioBrick this in the future, for now, we grew up cultures of our transposon and tested the ability of our FRED system to sense toxins. We didn't just want to sense toxins however, we wanted to be able to sense toxins in tailings ponds. to do this, we grew up our transposon clone in media, aspirated the media and then placed it in tailings pond water samples. Upon addition of our sugar-reporter conjugate, CPRG, we monitored the formation of CPR electrochemically, which would be indicative of LacZ production, indicating activity of our toxin sensory element. All our electrochemical protocols can be found here. The results of this assay can be shown below.</p><br />
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<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 />
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<h2> Taking FRED out to the field! </h2><br />
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<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to perform a kind of field test with FRED 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 />
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<p> We also created a video to show how we would test this water sample with our prototype and software package. This video can be found below.</p><br />
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<h2><u>Putting OSCAR together </u></h2><br />
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<h2> Putting our Killswitch into OSCAR - Can we use our Auxotroph with the Petrobrick?</h2><br />
<p><b>In fact 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 />
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</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 />
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<H2> Putting OSCAR into Action! </h2><br />
<p>Once we had tested FRED and shown that we could use him to detect toxins in tailings samples we wanted to put OSCAR into action in his home the bioreactor. By the end of the summer, we had designed and built a lab scale prototype of our bioreactor system. However, to better understand the needs of the oil sands industry we approached <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Kelly Roberge</a>, an oil sands consultant specializing in tailings ponds. Through speaking with Mr. Roberge, we were able to better understand the concerns that the oil sands industry has with the use and building synthetic biology systems to solve the challenges they face. In particular, Mr. Roberge had questions that surrounded the feasibility of scaling up our bioreactor to an industrial scale. As it turns out there are a number of considerations that should be made when moving from the lab scale to industrial scale. Particularly, because these transitions can be an imperfect when moving from the lab scale to industrial scale (>1000L tanks). Therefore we thought it would be important to test the feasibility of <b>using our bioreactor, belt skimmer, and Petrobrick, to demonstrate we can produce and isolate hydrocarbons</b>. These results are illustrated in the video below!</p><br />
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<b> Insert video here</b><br />
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<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be turn on this device to allow for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
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</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure X: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure X: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
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<p>With these experiments we have been able to demonstrate that both FRED and OSCAR are functional and can work on their respective applications even in the context of a large scale! By listening to professionals and bringing a <b>informed design</b> to our project we have been able to provide systems with real world applications. FRED can <b>detect compounds in tailings ponds</b> and we have been able to <b>scale up and optimize</b> OSCAR through our bioreactor and flux balance analysis work. Additionally, we have connected our projects together by providing a <b>double kill switch system </b> with both an auxotroph and inducible exonuclease system that increases the production of hydrocarbons in OSCAR! With these systems in place and a clear concept of the value of what our project has to offer, we look forward to seeing what the future holds for FRED and OSCAR!</p><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
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</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
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<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
<br />
</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
<br />
<br />
<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
<br />
<h3> GFP testing</h3><br />
<br />
</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
<br />
<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
<br />
<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
<br />
<h3> Results </h3><br />
<br />
<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
<br />
<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
<br />
<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
<br />
<h3> Kill Gene Testing </h3><br />
<br />
<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
<br />
<h3> Results </h3><br />
<br />
</html>[[File: 24 hour assay with mgtAp-mgtArb-S7 Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
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<br />
<h2>An alternative: a glucose repressible system</h2><br />
<br />
<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
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</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
<html><br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
<html><br />
<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
</html> [[File:Calgary_RhaGFPFinal.png|thumb|600px|centre|Figure ]] <html><br />
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<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T02:54:48Z<p>Rpgguardian: </p>
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<img src="https://static.igem.org/mediawiki/2012/8/8c/UCalgary2012_FRED_Killswitch_Regulation_Low-Res.png" style="float: right; padding: 10px; height: 200px;"></img><br />
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
<br />
</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
<br />
<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
<br />
<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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</html><br />
[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
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<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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</html><br />
[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
<br />
</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<br />
<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
<br />
<h3> GFP testing</h3><br />
<br />
</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
<br />
<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
<br />
<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
<br />
<h3> Results </h3><br />
<br />
<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
<br />
<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
<br />
<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
<br />
<h3> Kill Gene Testing </h3><br />
<br />
<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
<br />
<h3> Results </h3><br />
<br />
</html>[[File: 24 hour assay with mgtAp-mgtArb-S7 Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
<br />
<br />
<h2>An alternative: a glucose repressible system</h2><br />
<br />
<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
<html><br />
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</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
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<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
</html> [[File:Calgary_RhaGFPFinal.png|thumb|350px|centre|Figure ]] <html><br />
<br />
<br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/RegulationTeam:Calgary/Project/HumanPractices/Killswitch/Regulation2012-10-27T02:54:08Z<p>Rpgguardian: </p>
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<h2> Tight Regulation </h2><br />
<p>Inducible kill systems are not new to iGEM. Looking through the registry, there are several constructs such as the inducible BamHI system contributed by Berkley in 2007 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_I716462">BBa_I716462</a>) and <a href="http://partsregistry.org/Image:UoflBamHIdatasheet.png">tested by Lethbridge in 2011</a>. This uses a <i>BamHI</i> gene downsteam of an arabinose-inducible promoter. Another example is an IPTG inducible Colicin construct (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K117009">BBa_K117009</a>) submitted by NTU-Singapore in 2008. One major problem with these systems however is a lack of tight control. As was demonstrated by the Lethbridge 2011 team, this part has leaky expression when inducer compound is not present. The frequently used lacI promoter has similar problems when not used in conjunction with strong plasmid-mediated expression of lacI. This can be seen in our electrochemical characterization of the UidA hydrolase enzyme (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902002">BBa_K902002</a>) shown here. Tight control is not only a problem for kill switch application, but for any application requiring strict regulation. As such, we decided that expanding the registry repertoire of control elements would be useful for our system as well as a variety of other applications. Therefore we added a new level of regulation in addition to the promoter, a riboswitch</p><br />
<h2> Introducing the Riboswitch </h2><br />
<p>Riboswitches are small pieces of mRNA which bind ligands to modify translation of downstream genes. These sites are engineered into circuits by replacing traditional ribosome binding sites with riboswitches. The riboswitch is able to bind its respective ligand to inhibit or promote binding of translational machinery (Vitreschak <i>et al</i>, 2004). Riboswitches can be used in tandem with an appropriate promoter to enable tighter control of gene expression. Given this opportunity for control, and that ligands for riboswitches are often inexpensive small ions, these methods might be a feasible solution for controlling the kill switch in our industrial bioreactor.</p><br />
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</html> [[File:UofC_RIBOSWITCH.png|thumb|350px|centre|Figure 1: A simply diagram illustrating the riboswitch and the three metabolite, magnesium, manganese and molybdenum, we have tested.]] <html><br />
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<p>We explored 3 different riboswitches, each responsive to a different metabolite (magnesium, manganese or molybdate co-factor) that would be inexpensive to implement into a bioreactor environment. Additionally, we also investigated a repressible and inducible promoter, responsive to glucose and rhamnose respectively.</p><br />
<p>The general approach taken to build the system was constructing the promoter with the respective riboswitch followed by the kill genes. </p><br />
<h2>Magnesium riboswitch</h2><br />
<p>The magnesium riboswitch that we looked at is repressed in the presence of magnesium ions. This system has two control components – a promoter and a riboswitch. Normally the magnesium (mgtA) promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and the magnesium (mgtA) riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902009</a>) are activated if there is a deficiency of magnesium in the cell (Winnie and Groisman, 2010). The sequence of the <i>mgtA</i> promoter and riboswitch was obtained from Winnie and Groisman. A lack of magnesium activates other genes in <i>E. coli </i>to allow influx of magnesium into the cell. The two proteins in the cascade that activate the system are <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>) and <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>). <i>PhoQ</i> is the trans-membrane protein which gets activated in the absence of magnesium and phosphorylates <i>PhoP</i>. <i>PhoP</i> in turn binds to the mgtA promoter and transcribes genes downstream (Winnie and Groisman, 2010).</p><br />
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<h2>Manganese riboswitch</h2><br />
<p> Manganese is an essential micronutrient. It is an important co-factor for enzymes and it also reduces oxidative stress in the cell (Waters <i>et al</i>. 2011). Despite being an important micronutrient, it is toxic to cells at high levels. MntR protein detects the level of manganese in the cell and acts as a transcription factor to control the expression of manganese transporter such as MntH, MntP and MntABCDE. In order to regulate these genes <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) binds to the mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>). The manganese homeostasis is also controlled by the manganese riboswitch <i>mntPrb</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K90274</a>). The sequences of the <i>mntP</i> promoter and the <i>mntP</i> riboswitch was obtained from the Waters et al, 2011.</p><br />
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[[File:Ucalgary2012 KillswitchstuffsystemsAandB.png|thumb|800px|left|Figure 2: '''A)''' MgtA pathway in <i>E. coli</i>. <i>PhoQ</i> is the transmembrane receptor which, upon detecting low magnesium concentrations, phosphorylates <i>PhoP</i> which acts as a transcription factor, transcribing genes downstream of the MgtA promoter necessary for bringing magnesium into the cell. There is a second level of control with the magnesium riboswitch. In the presence of high magnesium the riboswitch forms a secondary structure which does not allow the ribosome to bind to the transcript, thus inhibiting translation. '''B)''' In the presence of manganese, the <i>MntR</i> protein represses the <i>mntH</i> transporter, preventing the movement of manganese and also upregulating the putative efflux pump. Genes downstream of the mntP promoter are thus transcribed in the presence of manganese. The addition of the <i>MntR</i> protein in this system allows for tighter regulation of the system.]]<html><br />
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<h2> The Moco Riboswitch </h2><br />
<br />
<p>The molybdenum cofactor riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>) is an RNA element which responds to the presence of the metabolite molybdenum cofactor (MOCO) (Regulski et al, 2008). This RNA element is located in the <i>E.coli</i> genome just upstream of the <i>moaABCDE</i> operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>), containing the moco synthesis genes. Moco is an important co-factor in many different enzymes. The moco riboswitch has 2 regions: an aptamer domain and the expression platform. When moco is present in the cell it will bind to the aptamer region in the riboswitch causing an allosteric change. This allosteric change affects the expression platform by physically hiding the ribosome binding site which prevents translation.</p><br />
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[[File:Moco_riboswitchCalgary2012.jpg|thumb|750px|center|Figure 3: This picture depicts the Moco RNA motif which is upstream of the <i>moaABCDE</i> operon. ]]<html> <br />
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<h2> Building the Systems </h2><br />
<br />
<p> Using these riboswitches, we wanted to design a system where we would place our kill genes downstream, and then supplement our bioreactor with the appropriate ions to keep the systems turned off. We biobricked and submitted DNA for the the <i>mgtaP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902009">BBa_K902009</a>) and mntP promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902073">BBa_K902073</a>) as well as their respective riboswitches (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902008">BBa_K902008</a>) (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902074">BBa_K902074</a>) and the moco riboswitch (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902023">BBa_K902023</a>). In addition, we also biobricked some of the regulatory proteins: <i>PhoP</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010">BBa_K902010</a>), <i>PhoQ</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011">BBa_K902011</a>), <i>mntR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902030">BBa_K902030</a>) and the Moa Operon (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902024">BBa_K902024</a>) . Our final system would inovolve constitutive expression of these necessary regulatory elements upstream of our riboswitches and kill genes. An example of the manganese system is shown in figure 4. </p><br />
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</html>[[File:U.Calgary.2012_10.02.2012_Final_Construct_1.png|thumb|600px|center|Figure 4: Final construct for the manganese system. The circuit includes a TetR promoter, RBS, mntR, double terminator, mntP promoter, mntP riboswitch, <i>S7</i>, mntP riboswitch and <i>CViAII</i>.]]<html><br />
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<a name="killswitch"></a><h2> Characterizing the riboswitches </h2><br />
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<h3> GFP testing</h3><br />
<br />
</html>[[File:MgtA circuits Ucalgary1.png|thumb|150px|right|Figure 5: In these set of circuits, <i>TetR</i>-RBS-K082003 serves as a positive control and the <i>mgtAp-mgtArb</i> serves as a negative control.]]<html><br />
<br />
<p> In order to test the control of these promoters and riboswitches, we constructed them independently and together upstream of GFP (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K082003">BBa_K082003</a>) with an LVA tag. Figure 5 shows these circuits for the mgtA system. Identical circuits were designed for all three systems, however only the top two were needed for the mocoriboswitch system.</p><br />
<br />
<p>We then tested the aforementioned circuits by growing cells containing our circuits with varying concentrations of their respective ions. Our detailed protocols can be found <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. We then measured fluorescent output, normalizing to a negative control not expressing GFP.</p><br />
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<h3> Results </h3><br />
<br />
<p>So far, we have been able to obtain results for our magnesium system, as can be seen in figure 6. </html><br />
[[File:Magmesium graph ucalgary2.png|thumb|500px|left|Figure 6: This graph represents the relative fluorescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html></p><br />
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<br />
<p>As the graph shows, there is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together as compared to the <i>mgtA</i> riboswitch alone under the control of TetR promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J13002">BBa_J13002</a>). This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that the magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p><br />
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<p> It is important to consider however that the control elements of the system, <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902010"><i>PhoP</i> </a> and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902011"> <i>PhoQ</i></a>, that were described above were not present in the circuits tested and therefore there is GFP expression in at the inhibitory concentration (10mM MgCl<sub>2</sub>). We believe that having the regulatory elements would give us better control and limit the leakiness.</p><br />
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<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium, as high as 120mM (Kim <i>et al</i>. 2011). As can be seen, this would inhibit the system. Therefore, if our bacteria were to escape into the tailings, the kill genes would not be activated and the bacteria would be able to survive. However, we feel that this could still be an incredibly useful system for other teams for both killswtitch and non-killswitch-related applications, making it still a valuable contribution to the registry. </p><br />
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<h3> Kill Gene Testing </h3><br />
<br />
<p> While building our systems with GFP in order to test their control, we also constructed them with our kill genes. This was delayed substantially however due to problems in their synthesis. Specifically, the micrococcal nuclease that arrived from IDT had a 1bp point mutation which changed an isoleucine residue into a lysine. Initially, our systems resulted in no killing of cells. Therefore we had to mutate this residue using <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mutagenesis"> site-directed mutagenesis</a>. Once completed, we were able to begin testing. With our GFP data collected, we moved on to characterizing the mgtA control system upstream of our <i>S7</i> kill gene (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902019">BBa_K902019</a>). To test the circuits, we incubated cells expressing our construct with varying concentrations of magnesium. We then measured both Colony Forming Units (CFU) and OD 600. For a deatiled protocol, see <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/mgtacircuit">here</a>.</p><br />
<br />
<h3> Results </h3><br />
<br />
</html>[[File: 24 hour assay with mgtAp-mgtArb-S7 Ucalgary.png|thumb|750px|center| Figure 7: This shows the OD600 values of mgtA circuits with S7 both mutated and unmutated. The negative control consists of <i>mgtAp-mgtArb</i>.]]<html><br />
<p> Figure 7 shows that the mgtAp-mgtArb-S7 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902018">BBa_K902018</a>) starts acting approximately 4 hours after induction. However, it also shows that 10mM MgCl<sub>2</sub> is not enough salt to inhibit the entire system because there is no difference in OD600 measurement at 4hr time point between 10mM and the 0mM concentrations. This test needs to be repeated with higher concentrations of Mg<sup>2+</sup> however this data suggests that the mutagenesis was successful and <i>S7</i> is active and killing the cells at approximately 4hr which does not necessarily reflect solely upon the activity of <i>S7</i> but also on the response time of the mgtA system.</p><br />
<br />
<br />
<h2>An alternative: a glucose repressible system</h2><br />
<br />
<p>Based on the problem with the magnesium system in relation to tailings pond conditions, we wanted to find an alternative. We found a promoter that was induced by rhamnose and repressed by glucose. This seemed to be a very suitable candidate for controlling the kill switch in the bioreactor since the promoter was shown to be tightly repressed by glucose. We could supplement the bioreactor with glucose to inhibit expression of the kill genes in the bioreactor. Escape of bacteria into the tailings ponds would cause expression of the kill genes due to lack of glucose in the surrounding environment.<br />
</p> <br />
<p>This promoter, known as <i>pRha</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>), is responsible for regulating genes related to rhamnose metabolism and contains a separate promoter on its leading and reverse strands (see figure 8). <i>RhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) and <i>RhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) serve to regulate expression of the rhamnose metabolism operon <i>rhaBAD</i>. The <i>RhaR</i> transcription factor is activated by L-rhamnose to up-regulate expression <i>rhaSR</i> operon. In turn, the resulting <i>RhaS</i> activates the <i>rhaBAD</i> operon to generate the rhamnose metabolism genes (Egan & Schleif, 1993).</p><br />
<br />
</html>[[File:NativeRhamnosePromoter_Calgary2012.jpg|thumb|750px|center|Figure 8: The rhamnose metabolism genes as they exist in Top Ten <i>E. coli</i>]]<br />
<html><br />
<br />
</html>[[File:PrhaFinal.png|thumb|750px|center|Figure 9: The rhamnose metabolism genes native to <i>E. coli</i>]]<br />
<html><br />
<p>Our kill system is different from the native rhamnose system with the <i>rhaR</i> and <i>rhaS</i> control genes. We have constitutively expressed <i>RhaS</i> to overcome dependency on rhamnose to cause activation of the kill switch. While <i>RhaS</i> is continuously present, the system is shut off in the presence of glucose. However, in the outside environment glucose levels are lower such that <i>RhaS</i> is able to activate the kill genes.</p><br />
<h3>Building the system</h3><br />
<p>Our team had <i>pRha</i> promoter (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902065">BBa_K902065</a>) commercially synthesized as per the sequence given by Jeske and Altenbuchner (2010). The <i>rhaS</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902068">BBa_K902068</a>) and <i>rhaR</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K902069">BBa_K902069</a>) genes were amplified via PCR from Top 10 <i>E. coli</i> using Kapa HiFi polymerase. </p><br />
<p>We tested the unoptimized rhamnose system using a fluorescent output. INSERT FIGURE </p><br />
<p>Additionally, we also tested the rhamnose system with micrococcal nuclease in the presence of glucose and rhamnose in both Top10 cells as well as glyA knockout from the Keio knockout collection. INSERT FIGURE AND DESCRIBE STUFF</p><br />
</html> [[File:RhaGFPFinal.png|thumb|350px|centre|Figure ]] <html><br />
<br />
<br />
<p><br />
<h2> The Glycine Auxotroph </h2><br />
<p> The idea of using an auxotropic system was initially considered, however due to the pricing of this system we felt it to be inappropriate for a large scale bioreactor. Auxotrophic systems that we had looked into included the 5-fluoro-orotic acid and histidine, which were both found to be expensive. This idea was reconsidered when our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Variability Analysis</a> showed that the Petrobrick system can be optimized with glycine added to the media. The production of hydrocarbons increased by a factor of 3 with our glycine media when compared to Washington’s production media. This finding justified our introduction of a glycine auxotrophic system as the increased efficiency of the Petrobrick in addition to another safety feature far outweighed the cost of implementing the system. This is feasible because glycine is not readily found in the environment and is relatively inexpensive to supplement on a large scale. </p> <p> We used a knockout strain JW2535-1 from the Keio collection in which the gene responsible for the synthesis of glycine was knocked out. The bacteria become dependent on glycine in the environment. The JW2535-1 knockout strain used works directly on glyA which is a component of the glycine hydroxymethyltransferase by mutating the glyA into Kan which overall prevents the bacteria’s growth. A glycine assay was set up to determine concentrations of glycine needed for the survival of the bacteria. The bacteria were grown on plate with glycine concentrations ranging from 1nM to 100mM. When zero glycine was added to the media there was some bacterial growth over time. This system will therefore need to work in conjunction with the kill switch system as another layer of security to reduce possibility of escapers. Please see our <a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy Page</a> for more information. </p><br />
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}}</div>Rpgguardianhttp://2012.igem.org/File:Calgary_RhaGFPFinal.pngFile:Calgary RhaGFPFinal.png2012-10-27T02:47:59Z<p>Rpgguardian: </p>
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<ul><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
</ul><br />
</li><br />
<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
<br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
</ul><br />
</html>|<br />
<br />
TITLE=Synergy: Putting it all Together|CONTENT=<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/03/UCalgary2012_FRED_and_OSCAR_Synergy.png" style="padding: 10px; float: right;"></img><br />
<h2>Incorporating Human Practices in the Design of our System </h2><br />
<p>In the earlier stages of our project, we realized that in order to give our project the best chance of being implemented, we needed to do it in a way that was in line with both industry’s wants and needs. To ensure that we did this, we established a dialogue with several experts in order to get their opinions on how we should approach our project. This led to an <b>informed design</b> of our system, in which we emphasized the need for both physical and genetic containment devices. </p><br />
<br />
<h2>Have we accomplished our goal?</h2><br />
<br />
<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’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 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> 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 />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:36:31Z<p>Rpgguardian: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<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 />
<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 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> 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 />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:29:34Z<p>Rpgguardian: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/OSCAR">OSCAR</a><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Decarboxylation">Decarboxylation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/CatecholDegradation">Decatecholization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis">Flux Analysis</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Bioreactor">Bioreactor</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Upgrading">Upgrading</a></li><ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Desulfurization">Desulfurization</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/Denitrogenation">Denitrogenation</a></li></ul> <br />
</ul><br />
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<li><a href="https://2012.igem.org/Team:Calgary/Project/Synergy">Synergy</a></li><br />
</li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/References">References</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Attributions">Attributions</a></li><br />
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TITLE=Synergy: Putting it all Together|CONTENT=<br />
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<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 />
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<h2>Have we accomplished our goal?</h2><br />
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<p>Nearing the end of our project however, we wanted to see if we had accomplished what we set out to do. So we decided to go back to the experts, this time taking the progress we’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 />
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<h2><u>Putting our Killswitch Together</u></h2><br />
<h2>Testing the Requirement of Glycine With our Auxotroph</h2><br />
<p>Our <a href="https://2012.igem.org/Team:Calgary/Project/OSCAR/FluxAnalysis"><b>flux-based analysis</b></a> allowed us to realize the potential for glycine to be used not only as a way to increase the yield of OSCAR, but also as an auxotrophic killswitch. This allowed our model to be used not only to inform our wetlab, but also our human practices. We wanted to see how this auxotrophic marker system could work with one of our inducible killswitch constructs. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
<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 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> 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 />
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<br />
<b> Insert video here</b><br />
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<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be turn on this device to allow for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
<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 />
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}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:26:28Z<p>Rpgguardian: </p>
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<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project">Overview</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/DataPage">Data Page</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/Accomplish">Accomplishments</a></li><br />
</ul><br />
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<li><br />
<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/HumanPractices">Human Practices</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Collaborations">Initiative</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Interviews">Interviews</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Design">Design</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Killswitch</a></li><ul><li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">Regulation</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">Kill Genes</a></li></ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Safety">Safety</a></li><br />
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<a class="drop" href="https://2012.igem.org/Team:Calgary/Project/FRED">FRED</a><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Detecting">Toxin Sensing</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Reporting">Electroreporting</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Modelling">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:Calgary/Project/FRED/Prototype">Device Prototype</a></li><br />
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<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 />
<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>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 />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our auxotroph with the Petrobrick?</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 />
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<br />
<br />
<br />
<br />
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<br />
}}</div>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T02:20:31Z<p>Rpgguardian: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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</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 />
<h2>Testing the Auxotrophic Marker as a Kill Switch</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 />
<b>INSERT BEAUTIFUL FIGURE HERE!!!!</b><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 />
<br />
<h2> Putting our Killswitch into OSCAR - Can we use our auxotroph with the Petrobrick?</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>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T01:40:01Z<p>Rpgguardian: </p>
<hr />
<div>{{Team:Calgary/MainHeader | <html><img src="https://static.igem.org/mediawiki/2012/8/82/UCalgary2012_Offical_Logo_Purple.png"></img></html>}}<br />
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<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> 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. 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>Rpgguardianhttp://2012.igem.org/Team:Calgary/Project/SynergyTeam:Calgary/Project/Synergy2012-10-27T01:38:48Z<p>Rpgguardian: </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 />
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</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> 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. We procured a Keio Knockout Collection Strain which deleted <i>glyA</i> an important enzyme in glycine metabolism making it auxotrophic for this compound. We wanted to identify the concentration of glycine required for its growth as shown below.<br />
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</html>[[File:Calgary GlycineKODeathAssay.png|thumb|500px|center|Figure 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 />
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As identified by the growth assay, the glycine knockout is not capable of completely preventing growth of the strain even at very low concentrations of glycine. This identifies that it is important to continue to use our kill switch mechanism in combination with the auxotroph to control the cells. Now, with the concentrations ideal for glycine growth determined, we transformed our rhamnose inducible killswitch construct containing S7 <b>(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902084">BBa_K902084</a>)</b> into our glycine knockout strain and attempted to characterize cell death over a variety of conditions.</p><br />
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<b>INSERT BEAUTIFUL FIGURE HERE!!!!</b><br />
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<h2> Putting our Killswitch into OSCAR</h2><br />
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<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 />
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<h2> Taking FRED out to the field! </h2><br />
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<p> Once we knew that we had a promoter/reporter system that could actually detect toxins found in tailings ponds within the laboratory, the next challenge was to detect tailings pond toxins with our FRED prototype on site. Unfortunately, there are very strict regulations surrounding tailings ponds, and the publication of information pertaining to their contents. As such, obtaining permissions for a tailing pond field test was not possible within the time frame of our project. Because we did want to 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 />
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<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 />
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<p>In short, the bioreactor was fillwed with 50:50 LB:Washington Production Media and we allowed the Petrobrick to grow over a 72 hour period. Afterwards, we demonstrated how our belt skimmer could be turn on this device to allow for removal of the hydrocarbons. Because the hydrocarbons need to be extracted, we added ethyl acetate to allow for extraction, and demonstrated that our belt skimmer could selectively pick up the organic layer. Finally we ensured that this organic phase contained hydrocarbons by running this segment on the GC/MS as illustrated below.</p><br />
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</html>[[File:Calgary BioreactorValidation.png|thumb|500px|center|Figure X: The GC chromatograph from the solvent layer which was selectively used with the belt skimmer. A large peak was observed much greater than any of the others, suggesting that hydrocarbons were being selectively removed with the belt skimmer.]]<html><br />
</html>[[File:Calgary BioreactorValidationMS.png|thumb|300px|center|Figure X: MS data for the peak with a retention time of 12.7 min. The spectra suggests that the compound is a C16 hyrocarbon, validating that the upscaled bioreactor/belt skimmer combination can be used to isolate hydrocarbons.]]<html><br />
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<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 />
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<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 />
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</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 />
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}}</div>Rpgguardianhttp://2012.igem.org/File:Calgary_GlycineKODeathAssay.pngFile:Calgary GlycineKODeathAssay.png2012-10-27T01:32:43Z<p>Rpgguardian: </p>
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<div></div>Rpgguardian