Difference between revisions of "Team:NAIT Edmonton/Parts"

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{{NAIT_Edmonton}}
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<h2> Part Documentation</h2>
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<head>
  
<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
 
<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
 
  
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<title>Team NAIT 2015</title>
  
<div class="highlightBox">
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<link href='http://fonts.googleapis.com/css?family=Source+Sans+Pro' rel='stylesheet' type='text/css'>
<h4>Note</h4>
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<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script>
<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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<script type="text/javascript" src="https://2015.igem.org/Team:NAIT_Edmonton/Results/PrefixTree.js?action=raw&amp;ctype=text/javascript"></script>
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<!-----------https://static.igem.org/mediawiki/2015/d/d8/El_Capitan_.ttf------------------->
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<script type="text/javascript">
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$(document).ready(function() {
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// grab the initial top offset of the navigation
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  var stickyNavTop = $('.nav').offset().top;
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  // our function that decides weather the navigation bar should have "fixed" css position or not.
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  var stickyNav = function(){
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    var scrollTop = $(window).scrollTop(); // our current vertical position from the top
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    // if we've scrolled more than the navigation, change its position to fixed to stick to top,
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    // otherwise change it back to relative
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    if (scrollTop > stickyNavTop) {
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        $('.nav').addClass('sticky');
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    } else {
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        $('.nav').removeClass('sticky');
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    }
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};
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stickyNav();
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// and run it again every time you scroll
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$(window).scroll(function() {
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stickyNav();
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});
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});
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</script>
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<script type="text/javascript" src="https://2015.igem.org/Team:NAIT_Edmonton/StickyMenu?action=raw&amp;ctype=text/javascript"></script>
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</head>
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<body>
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<div class="header">
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<div class="header_c">
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<a href="https://2015.igem.org/Team:NAIT_Edmonton"><img style="margin-top:15px;" src="https://static.igem.org/mediawiki/2015/2/2f/NAIT_Imagine.png" height="95px"></a>
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</div>
 
</div>
 
</div>
  
  
  
<h4>Adding parts to the registry</h4>
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<div class="nav">
<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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<div tabindex="0" class="onclick-menu">
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    <ul class="onclick-menu-content" style="list-style:none;">
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        <li><a href="https://2015.igem.org/Team:NAIT_Edmonton">home</a></li>
 +
        <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Team">team</a></li>
 +
        <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Project">project</a></li>
 +
        <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Human">human practices</a></li>
 +
        <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Notebook">notebook</a></li>
 +
    </ul>
 +
</div>
  
<h4>What information do I need to start putting my parts on the Registry?</h4>
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<p>The information needed to initially create a part on the Registry is:</p>
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<div class="navigation">
 
<ul>
 
<ul>
<li>Part Name</li>
 
<li>Part type</li>
 
<li>Creator</li>
 
<li>Sequence</li>
 
<li>Short Description (60 characters on what the DNA does)</li>
 
<li>Long Description (Longer description of what the DNA does)</li>
 
<li>Design considerations</li>
 
</ul>
 
  
<p>
 
We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
 
  
 +
                <li><a href="https://2015.igem.org/Team:NAIT_Edmonton"><img
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                src="https://static.igem.org/mediawiki/2015/a/a9/NAIT_home.png" width="25px"></a></li>
  
 +
                <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Team">team</a>
 +
                    <ul>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Bios">bios</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Attributions">attributions</a></li>
 +
                      <li><a href="https://igem.org/Team.cgi?id=1787">official team profile</a>
 +
                    </ul>
 +
                </li>
  
 +
                <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Project">project</a>
 +
                    <ul>
 +
                    <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Desc">description</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Protocols">experiment and protocols</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Results">parts and results</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Modeling">modeling</a></li>
 +
                    </ul>
 +
                </li>
  
 +
                <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Human">human practices</a>
 +
                <ul>
 +
                    <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Outreach">community outreach</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Practices">policy and practices</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Collaborations">collaborations</a></li>
 +
                     
 +
                    </ul>
 +
                </li>
  
 +
                <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Notebook">notebook</a>
 +
                <ul>
 +
                    <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Safety">lab safety</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Achievements">achievements</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:NAIT_Edmonton/Logbook">log book</a></li>
 +
                </ul>
 +
                </li>
  
<h4>Inspiration</h4>
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            </ul>
<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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</div>
<ul>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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</ul>
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 +
</div>
  
  
<h4>Part Table </h4>
 
</html>
 
<groupparts>iGEM015 Example</groupparts>
 
<html>
 
  
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<div id="wrap">
  
  
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<style type="text/css">
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.top_slogan {text-align:center; font-family: 'Source Sans Pro', sans-serif; color:#0D4D8C; font-size:30px; padding: 40px 0px 40px 0px; font-style:strong; line-height:40px;}
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</style>
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<center><div class="top_slogan">Parts and Results</div></center>
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<div class="main_content">
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<center><img src="https://static.igem.org/mediawiki/2015/6/61/NAIT_p5.png" width="500px"></center>
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<p>The sequences that were ordered had a design flaw on them. The restriction sites were not on the correct place and were incompatible with pSB1C3. In order to fix them, we decided to create primers that would insert a point mutation in the restriction sites and, at the same time, add the correct iGEM prefix and suffix to the sequences.</p><br>
 +
 +
<p><img src="https://static.igem.org/mediawiki/2015/1/17/NAIT_p2.png" width="400px">A gradient PCR was conducted in order to determine the best annealing temperature (Ta) for the primers. It was observed that Ta=61ºC was optimum. A new PCR comprising all our sequences was done and, after using a PCR Purification kit, an agarose gel showed that out of 23 sequences, we were able to purify 18 (see Figure 2). Nonetheless, one of the sequences contained an illegal restriction site within itself and it was discarded.</p><br>
 +
 +
<p>The sequences, now containing an iGEM compatible prefix and suffix, needed a vector and a host organism so as to be expressed. T7 expression systems can produce a high copy of a Protein of Interest (POI), provided that the DNA sequence that encodes for the polypeptide is next to a T7 promoter. With regards to the vector, it was noted that BioBrick K1321338 (BBa_ K1321338), found on the iGEM 2015 Kit Plate #5, contained a T7 promoter and a Ribosome Binding Site. And pertaining the host organism, BL21 (DE3), a T7 E.coli expression strain was chosen as the system to synthesize our POIs</p><br>
 +
 +
<center><img src="https://static.igem.org/mediawiki/2015/e/e7/NAIT_p3.png" width="500px"></center>
 +
 +
<p>After transforming cells with BBa_K1321338, obtaining the plasmid (i.e., pSB1C3 with the BioBrick. Note: pSB1C3 contains a chloramphenicol resistant gene) via a MiniPrep Kit, and confirming its presence by running a sample in an agarose gel, a digestion with SpeI and PstI, and subsequent dephosphorylation was conducted so as to linearize the plasmid and prepare it for ligation to the sequences. Simultaneously, the sequences were digested with XbaI and PstI (see Figure 3).</p>
 +
 +
 +
<center><img src="https://static.igem.org/mediawiki/2015/4/43/NAIT_p4.png" width="500px"></center>
 +
 +
 +
<p>Ligation was performed using T4 DNA Ligase; subsequently, E. coli BL21 (DE3) was transformed with the ligation products and plated in LB + chloramphenicol agar. 34 plates (two per sequence of interest) were left 24 hours at 37ºC to ensure optimum growth of transformed cells. Grown colonies were accounted for and numbered (see Figure 4).</p><br>
 +
 +
<p>pSB1C3 Forward and Reverse Primers were used to confirm that the colonies actually contained the sequence. A gradient PCR showed that the optimum Ta was 57ºC, so a colony PCR was made following such parameter and an agarose gel was done to verify outcome of the PCR.</p><br>
 +
 +
<p>Data showed that, out of three hundred and two colonies, only five contained an insert (see Figure 5). Other colonies contained the plasmid without an insertion. This was concluded because the Forward and Reverse Primers amplify a fragment 339 bp long. The divergent colonies amplified longer fragments and, out of those six, only two had the size that was expected. These were sequences ‘Cys-Thr’ and ‘MLAB’ (see Figure 6, this figure only shows significant gels).</p><br>
 +
 +
 +
 +
<center><img src="https://static.igem.org/mediawiki/2015/9/97/NAIT_p6.png" width="500px"></center>
 +
 +
<p>These five positive colonies were cultured in 200mL of LB media with chloramphenicol and induced with IPTG to increase protein expression. The cells were pelleted and proteins were isolated using a NiNTA Kit.
 +
The samples were ran in a SDS-PAGE (see Figure 6). Only the sample containing the protein encoded by sequence ‘Cys-Thr’ showed bands. Additionally, the bands were ~19kDa in size, the expected size for this particular protein.
 +
</p><br>
 +
 +
 +
<p>Preliminary data shows that this engineered protein is able to show a different colouration after silver staining (see Figure 8)</p><br>
 +
 +
<center>
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<img src="https://static.igem.org/mediawiki/2015/1/11/NAIT_p7.png" width="100%"></center>
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<div class="clear"></div>
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</div>
 
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Revision as of 03:43, 19 September 2015

Team NAIT 2015

Parts and Results

The sequences that were ordered had a design flaw on them. The restriction sites were not on the correct place and were incompatible with pSB1C3. In order to fix them, we decided to create primers that would insert a point mutation in the restriction sites and, at the same time, add the correct iGEM prefix and suffix to the sequences.


A gradient PCR was conducted in order to determine the best annealing temperature (Ta) for the primers. It was observed that Ta=61ºC was optimum. A new PCR comprising all our sequences was done and, after using a PCR Purification kit, an agarose gel showed that out of 23 sequences, we were able to purify 18 (see Figure 2). Nonetheless, one of the sequences contained an illegal restriction site within itself and it was discarded.


The sequences, now containing an iGEM compatible prefix and suffix, needed a vector and a host organism so as to be expressed. T7 expression systems can produce a high copy of a Protein of Interest (POI), provided that the DNA sequence that encodes for the polypeptide is next to a T7 promoter. With regards to the vector, it was noted that BioBrick K1321338 (BBa_ K1321338), found on the iGEM 2015 Kit Plate #5, contained a T7 promoter and a Ribosome Binding Site. And pertaining the host organism, BL21 (DE3), a T7 E.coli expression strain was chosen as the system to synthesize our POIs


After transforming cells with BBa_K1321338, obtaining the plasmid (i.e., pSB1C3 with the BioBrick. Note: pSB1C3 contains a chloramphenicol resistant gene) via a MiniPrep Kit, and confirming its presence by running a sample in an agarose gel, a digestion with SpeI and PstI, and subsequent dephosphorylation was conducted so as to linearize the plasmid and prepare it for ligation to the sequences. Simultaneously, the sequences were digested with XbaI and PstI (see Figure 3).

Ligation was performed using T4 DNA Ligase; subsequently, E. coli BL21 (DE3) was transformed with the ligation products and plated in LB + chloramphenicol agar. 34 plates (two per sequence of interest) were left 24 hours at 37ºC to ensure optimum growth of transformed cells. Grown colonies were accounted for and numbered (see Figure 4).


pSB1C3 Forward and Reverse Primers were used to confirm that the colonies actually contained the sequence. A gradient PCR showed that the optimum Ta was 57ºC, so a colony PCR was made following such parameter and an agarose gel was done to verify outcome of the PCR.


Data showed that, out of three hundred and two colonies, only five contained an insert (see Figure 5). Other colonies contained the plasmid without an insertion. This was concluded because the Forward and Reverse Primers amplify a fragment 339 bp long. The divergent colonies amplified longer fragments and, out of those six, only two had the size that was expected. These were sequences ‘Cys-Thr’ and ‘MLAB’ (see Figure 6, this figure only shows significant gels).


These five positive colonies were cultured in 200mL of LB media with chloramphenicol and induced with IPTG to increase protein expression. The cells were pelleted and proteins were isolated using a NiNTA Kit. The samples were ran in a SDS-PAGE (see Figure 6). Only the sample containing the protein encoded by sequence ‘Cys-Thr’ showed bands. Additionally, the bands were ~19kDa in size, the expected size for this particular protein.


Preliminary data shows that this engineered protein is able to show a different colouration after silver staining (see Figure 8)