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| | + | {{Team:Calgary/Main}} |
| | <html> | | <html> |
| - | <div id="Banner"><h1>Raleway</h1></div> | + | |
| - | </html> | + | <section id="Content"> |
| - | {{Team:Calgary/ContentPage}}
| + | |
| - | <html> | + | <h1>Quantitative App</h1> |
| - | <script> | + | |
| - | $("a.inpage").click(function (e) {
| + | <h2>DNA binding to TALEs<h2> |
| - | e.preventDefault();
| + | |
| - | var targetpos = $($(this).attr('href')).position(); // note this line
| + | <p>The positive DNA's are colored red, negative DNA is green, TALEs are black and after binding (i.e. saturation) they turn blue. The sliders under "global settings" are parameters needed before the simulation runs. Given that a simulation can take 30 seconds to complete, they should not be changed frequently as it will take longer to show the results. The sliders under "display settings" are parameters needed after the simulation runs. Their responses are much faster and are best played by clicking on the plus sign to the right and hit "Play". If you only see a grey box, you may need to install the Mathematica CDF Player. The download link can be found <a href="http://www.wolfram.com/cdf-player/">here</a>. Otherwise, the data from our model, as well as images of the app we designed, can be found on the <a href="https://2013.igem.org/Team:Calgary/Project/OurSensor/Modelling/QuantitativeModelling">quantitative modelling page</a>. |
| - | var scrollpos = targetpos.top - 100;
| + | </p> |
| - | window.scrollTo(0,scrollpos);
| + | |
| - | }
| + | <img align="right" style="width:35%;" src="https://static.igem.org/mediawiki/2013/6/6b/UCalgary2013SWAgenModelInstruction.jpg" style="float: left; clear: both;"></img> |
| | + | |
| | + | <script type="text/javascript" src="http://www.wolfram.com/cdf-player/plugin/v2.1/cdfplugin.js"></script> |
| | + | <script type="text/javascript"> |
| | + | var cdf = new cdfplugin(); |
| | + | cdf.embed('http://igem.ucalgary.ca/misc/Deployed.cdf', 430, 800); |
| | + | </script> |
| | + | |
| | + | <h2>Enzyme kinetics of horseradish peroxidase</h2> |
| | + | |
| | + | <script type="text/javascript" src="http://www.wolfram.com/cdf-player/plugin/v2.1/cdfplugin.js"></script> |
| | + | <script type="text/javascript"> |
| | + | var cdf = new cdfplugin(); |
| | + | cdf.embed('http://igem.ucalgary.ca/misc/HorseradishPeroxidase.cdf', 350, 360); |
| | + | </script> |
| | + | |
| | + | <h2>Enzyme kinetics of beta-galactosidase</h2> |
| | + | |
| | + | <script type="text/javascript" src="http://www.wolfram.com/cdf-player/plugin/v2.1/cdfplugin.js"></script> |
| | + | <script type="text/javascript"> |
| | + | var cdf = new cdfplugin(); |
| | + | cdf.embed('http://igem.ucalgary.ca/misc/Beta-Galactosidase.cdf', 350, 330); |
| | </script> | | </script> |
| | | | |
| - | <section id="Content">
| + | <h2>Frequently Asked Questions |
| - | <h1>Detector Journal</h1>
| + | </h2> |
| - | Click <a href="#TestLink">here</a> to read chapter 4.
| + | |
| - | <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>
| + | |
| - | <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>
| + | |
| - | <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>
| + | |
| | | | |
| - | <h2>Week 2: May 6 - May 10</h2>
| + | <h3>1. Can I see your code? |
| - | <h3>next header</h3>
| + | </h3> |
| - | <h4>Level 4 Header</h4>
| + | <p>Sure! Here are <a href="https://static.igem.org/mediawiki/2013/3/3a/UCalgary2013SWTheCode.txt" target="_blank">DNA binding to TALEs agent based model</a>, |
| - | <p>This week we attended a workshop based on general molecular biology techniques.</p>
| + | <a href="https://static.igem.org/mediawiki/2013/b/b5/UCalgary2013SWHorseradishPeroxidaseCode.txt" target="_blank">horseradish peroxidase kinetic model</a>, and |
| | + | <a href="https://static.igem.org/mediawiki/2013/0/08/UCalgary2013SWBeta-GalactosidaseCode.txt " target="_blank">beta-galactosidase kinetic model</a>. |
| | + | </p> |
| | | | |
| - | <h2>Week 3: May 13 - May 17</h2>
| + | <h3>2. I see a block of text with red background. What should I do? |
| - | <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>
| + | </h3> |
| - | <a name="TestLink"></a>
| + | <p>Don't worry, everything is under control. For the DNA binding to TALEs agent based model, wait for at least 30 seconds. If the problem persists, reset the current time to 1. For the kinetic models, use the plus/minus sign to change the parameters, or simply hit auto-run on the upper-right corner. |
| - | <h2>Week 4: May 20 - May 24</h2>
| + | </p> |
| - | <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> | + | |
| - | <p>We learned about three very important TALE characteristics:
| + | |
| - | 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>
| + | |
| - | <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>
| + | |
| | | | |
| - | <h2>Week 5: May 27 - May 31</h2>
| + | </section> |
| - | <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>
| + | |
| - | </section>
| + | |
| | </html> | | </html> |
Quantitative App
DNA binding to TALEs
The positive DNA's are colored red, negative DNA is green, TALEs are black and after binding (i.e. saturation) they turn blue. The sliders under "global settings" are parameters needed before the simulation runs. Given that a simulation can take 30 seconds to complete, they should not be changed frequently as it will take longer to show the results. The sliders under "display settings" are parameters needed after the simulation runs. Their responses are much faster and are best played by clicking on the plus sign to the right and hit "Play". If you only see a grey box, you may need to install the Mathematica CDF Player. The download link can be found here. Otherwise, the data from our model, as well as images of the app we designed, can be found on the quantitative modelling page.
Enzyme kinetics of horseradish peroxidase
Enzyme kinetics of beta-galactosidase
Frequently Asked Questions
1. Can I see your code?
Sure! Here are DNA binding to TALEs agent based model,
horseradish peroxidase kinetic model, and
beta-galactosidase kinetic model.
2. I see a block of text with red background. What should I do?
Don't worry, everything is under control. For the DNA binding to TALEs agent based model, wait for at least 30 seconds. If the problem persists, reset the current time to 1. For the kinetic models, use the plus/minus sign to change the parameters, or simply hit auto-run on the upper-right corner.
"
