Team:Calgary/TestPage

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<div id="Banner"><h1>Raleway</h1></div>
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<h1>Detector Journal</h1>
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Click <a href="#TestLink">here</a> to read chapter 4.
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<h2><span class="blue">Week</span> 1: <span class="green">May 1</span> - <span class="orange">May</span> 3 <span class="yellow">bla bla bla</span></h2>
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<p>bla bla bla <span class="Blue">blue</span> bla bla bla <span class="Green">green</span> bla bla bla <span class="Orange">orange</span> bla bla bla <span class="Yellow">yellow</span> bla bla bla.</p>
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<p>This week we attended courses required by our University to work in the lab. Besides that, the undergraduate supervisors presented about the principles of Genetics and Synthetic Biology to the new team members.</p>
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<h2>Week 2: May 6 - May 10</h2>
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<p>This week we attended a workshop based on general molecular biology techniques.</p>
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<h2>Week 3: May 13 - May 17</h2>
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<p>We continued the molecular biology workshop. Also, the team members were assigned in different parts of the project such as TALEs as our detector, Ferritin as our reporter, Linker, Human Practices, Modelling and Business.</p>
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<h2>Week 4: May 20 - May 24</h2>
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<p>We did our literature search for optimization of TALE binding affinity. There were conflicting results for the optimal truncation of the N and C terminus. On N terminus, 152 (Miller et al., 2011), 147 (Mussolini et al., 2011), 186 (Slovenia’s TALEs), and 158 or 186 (Mercer et al., 2012) residues were retained. On C terminus, 28 or 63 (Miller et al., 2011), and 76 (Slovenia’s TALEs) residues were retained. </p>
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              <p>We learned about three very important TALE characteristics:
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Firstly, TALEs demonstrate polarity. In other words, they interact more strongly with DNA at the N terminus compared to the C terminus (Meckler et al., 2013). Secondly, the Thymidine nucleotide at the zero repeat is very important to the binding affinity of the TALE (Meckler et al., 2013). Thirdly, the binding affinity of TALE varies based on the types of the nucleotides present in its target site. The relative affinities are: NG(1)>NN (0.18)!HD (0.16) " NI (0.0016)>NK (0.00016) (Meckler et al., 2013). All these points must be carefully considered while designing the TALE to maximize its binding affinity and specificity. </p>
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                <p>The four TALEs designed by Slovenia 2012 iGEM team, were ordered from the registry (TALE A (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K782004" >BBa_K782004</a>), TALE B (<a href="http://parts.igem.org/Part:BBa_K782006" >BBa_K782006</a>) and TALE D (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K782005" >BBa_K782005</a>)). Primers were designed to incorporate the the target sequences of TALE A (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K782004" >BBa_K782004</a>) and TALE B (<a href="http://parts.igem.org/Part:BBa_K782006" >BBa_K782006</a>) ([A] and [B]) in the RFP generator. This plasmid will be used as a target sequence. Primers were also designed that incorporate selected point mutations of [A] and [B] into a biobrick. Once these plasmids are made they will be used to determine how the TALEs affinity is altered by mutations. In addition, two other TALEs were ordered from the the authors of the Meckler et al. 2013 paper.</p>
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<h2>Week 5: May 27 - May 31</h2>
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<p>We continued our literature search for a unique and stable pathogenic <i>E. coli</i> marker for the TALEs to bind to. Potential pathogenic O157:H7 <i>E. coli</i> markers: <i>stx1</i>, <i>stx2</i> (Yoshitomi et al., 2012), <i>uidA</i> (Yoshitomi et al., 2003 and Feng et al., 1994), <i>eaeA</i>, <i>hly</i>, <i>rbfE</i> (Fortin et al., 2000), and <i>Z3276</i> (Li & Chen, 2012). However, BLAST searches determined that the use of only one 18 bp sequence is not sufficiently specific and sensitive to detect pathogenic markers. Should we continue to search for only one marker or more than one or expand the detection to any toxic Shiga producing strains?</p><p>In addition to searching for a pathogenic <i>E. coli</i> marker, we also started working on the TALE sequence and its construction. The TALE sequence chosen has a truncation in both N- and C-terminus. The truncated N-terminus has 111 residues and the C-terminus has 42 residues (Meckler et al., 2013). The sequence was codon optimized for <i>E.coli</i>. <a href="http://tools.neb.com/NEBcutter2/" >NEBCutter 2.0</a> was used to screen restriction sites for PstI, EcoRI, NotI, XbaI, SpeI, NgoMIV and AgeI, all present in the RFC 25. Cut sites for BsaI and Esp3I were also removed to pave the road for Golden Gate Assembly method. The original TALE sequence has 11 cut sites: BsaI (3), PstI (1), EcoRI (1), Esp3I (4), AgeI (2). The restriction sites were then eliminated with silent mutations, based on a <i>E. coli</i> codon usage table (San Diego State University, 2012 modified from Maloy, Stewart & Taylor, 1996).</p><p>The large size of the TALE prevents simple cloning, therefore we looked to Golden Gate Assembly. Golden Gate Assembly is a subcloning strategy that uses type IIs restriction enzymes, which cut outside of their recognition sequence allowing ligation into a final product that lacks any specific scar sites (Engler, Kandzia & Marillonnet, 2008).</p>
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<li id="Link0" class="SlideLink">Intro</li><li id="Link1" class="SlideLink">Problem</li><li id="Link2" class="SlideLink">Situation</li><li id="Link3" class="SlideLink">Idea</li><li id="Link4" class="SlideLink">Solution</li><li id="Link5" class="SlideLink">Platform</li>
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<h2>Our Project</h2>
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Outbreaks of foodborne illnesses are a growing problem in our lives. In 2011, the Centers for Disease Control and Prevention (CDC) in the United States, identified 767 outbreaks affecting nearly 14,000 people of foodborne illnesses. Of these, pathogenic <i>E. coli</i> was a recurring theme in many of these outbreaks. In Alberta, we recently experienced our own foodborne disease outbreak in late 2012. This outbreak was the result of pathogenic <i>E. coli</i> serotype O157 and led to significant food recall alongside many hospitalizations, deaths, massive economic losses and an overall loss of consumer confidence in food safety. Current detection methods require long incubation times to amplify <i>E. coli</i> in the sample and followed by amplification to verify the presence of known genes that are associated with pathogenic <i>E. coli</i>.
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One of the contributing factors connected with the outbreak in Alberta was the lack of rapid on-site detection systems available. Thus, the University of Calgary 2013 iGEM Collegiate team is using synthetic biology to develop system to rapidly detect the presence of pathogenic <i>E. coli</i> in the beef industry. By using engineered biological nanoparticles and DNA binding proteins we can specifically detect pathogenic DNA sequences. Our biosensor functions at the genomic level to detect the presence or absence of pathogenic <i>E. coli</i> in a given sample. This system allows us to pinpoint contamination during meat processing and also provides the ability to prescreen cattle in a preventative way to limit potential sources of contamination from the processing chain. Our system provides a powerful new tool for food safety, but also shows promise as a platform for the rapid detection of target organisms that are identified as key targets in a myriad of sectors from health to environment to biosecurity.
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<a href="https://2013.igem.org/Team:Calgary/Project/OurSensor">
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<h2>Our Sensor</h2>
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<img src="https://static.igem.org/mediawiki/2013/4/44/Calgary2013_Our_Sensor.png">
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<p>Check out what we did in the lab this year in response to <i>E. coli</i> contamination in the beef industry. Learn about the design of our detector, linker, reporter as well as the development of a prototype and mathematical model.</p>
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<h2>Data Page</h2>
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<p>Want to see a summary of what we accomplished this season? Click here to check out our data page where we outline all of the work that we’ve done to date!</p>
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<a href="https://2013.igem.org/Team:Calgary/Project/HumanPractices">
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<h2>Human Practices</h2>
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<img src="https://static.igem.org/mediawiki/2013/5/54/Calgary2013_Human_Practices.png">
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<p>Check out how Human Practices helped to guide the development of our sensor. Learn how we spent time talking to various experts in the beef industry in order to design our project with our end-user in mind.</p>
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<a href="https://2013.igem.org/Team:Calgary/Project/Collaboration">
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<h2>Collaboration</h2>
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<p>We worked hard with the Paris Bettencourt team this season to develop useful tools for the rest of the iGEM community. Click here to find out what our collaboration can add to iGEM. </p>
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Revision as of 22:04, 27 September 2013

Our Project

Outbreaks of foodborne illnesses are a growing problem in our lives. In 2011, the Centers for Disease Control and Prevention (CDC) in the United States, identified 767 outbreaks affecting nearly 14,000 people of foodborne illnesses. Of these, pathogenic E. coli was a recurring theme in many of these outbreaks. In Alberta, we recently experienced our own foodborne disease outbreak in late 2012. This outbreak was the result of pathogenic E. coli serotype O157 and led to significant food recall alongside many hospitalizations, deaths, massive economic losses and an overall loss of consumer confidence in food safety. Current detection methods require long incubation times to amplify E. coli in the sample and followed by amplification to verify the presence of known genes that are associated with pathogenic E. coli.

One of the contributing factors connected with the outbreak in Alberta was the lack of rapid on-site detection systems available. Thus, the University of Calgary 2013 iGEM Collegiate team is using synthetic biology to develop system to rapidly detect the presence of pathogenic E. coli in the beef industry. By using engineered biological nanoparticles and DNA binding proteins we can specifically detect pathogenic DNA sequences. Our biosensor functions at the genomic level to detect the presence or absence of pathogenic E. coli in a given sample. This system allows us to pinpoint contamination during meat processing and also provides the ability to prescreen cattle in a preventative way to limit potential sources of contamination from the processing chain. Our system provides a powerful new tool for food safety, but also shows promise as a platform for the rapid detection of target organisms that are identified as key targets in a myriad of sectors from health to environment to biosecurity.

Retrieved from "http://2013.igem.org/Team:Calgary/TestPage"