Team:Alberta/Protocols

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     <div class="titlebar">
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<br>
<br>
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      <h3><font color="#A80000"><font size="6">The Littlest Mapmaker</font></font></h3>
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<img src="/wiki/images/4/41/Ab_mapmen_title.png" width="600" height="95"></img><img src="/wiki/images/a/a5/2013-igem-logo.png" width="198" height="95"></img>
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      <h4><i><font size="2">"Exploration into the world of <font color="#A80000">DNA Computing</font>"<br/>
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        Team Alberta: University of Alberta</font></i></h4>
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      <a href="http://www.ualberta.ca" class="ualberta-logo"><img src="/wiki/images/b/b3/Ualberta-logo.png" alt="University of Alberta"></img></a>
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      <a href="https://2013.igem.org/Team:Alberta" class="igem-logo"><img src="/wiki/images/a/a5/2013-igem-logo.png" alt="iGem Main Page"></img></a>
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     </div>
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     <div class="bin">
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             <li><a href="/Team:Alberta/Parts">Submitted Parts</a></li>
             <li><a href="/Team:Alberta/Parts">Submitted Parts</a></li>
             <li><a href="/Team:Alberta/Accomplishments">Accomplishments</a></li>
             <li><a href="/Team:Alberta/Accomplishments">Accomplishments</a></li>
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            <li><a href="/Team:Alberta/Attributions">Attributions</a></li>
 
           </ul>
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             <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Alberta">Official Profile</a></li>
             <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Alberta">Official Profile</a></li>
             <li><a href="/Team:Alberta/Sponsors">Sponsors</a></li>
             <li><a href="/Team:Alberta/Sponsors">Sponsors</a></li>
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            <li><a href="/Team:Alberta/Attributions">Attributions</a></li>
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       </ul>
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       <div class="sidebar">
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         <div class="sidebar_block">
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           <span class="saying">Welcome to the Team Alberta Wiki!
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          <h5>Places</h5>
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             <div id="box" style="width: 450px; padding: 5px; border: 3px solid #000; background-color: #000000;">
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          <a href="#Top"><p>Top</p></a>
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             <div id="template" style="text-align: center; font-weight: bold; font-size: large; color: #f6f6f6; padding: 5px;">
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          <a href="#Parts"><p>The Parts</p></a>
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              For visitors: this site is currently under construction. Please contact our Student Liason, Dawson at                  daocun@ualberta.ca, for more information on our current project and how to support us!
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           <a href="#Build"><p>Building the Plasmids</p></a>
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             </div>
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          <h5>Project Sections</h5>
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             </div>
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            <a href="/Team:Alberta/Background"><p>Background</p></a>
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          </span>
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             <a href="/Team:Alberta/Overview"><p>Overview</p></a>
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            <a href="/Team:Alberta/Results"><p>Results</p></a>
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             <a href="/Team:Alberta/Protocols" class="active"><p>Protocols</p></a>
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             <a href="/Team:Alberta/Parts"><p>Submitted Parts</p></a>
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             <a href="/Team:Alberta/Accomplishments"><p>Accomplishments</p></a>
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       <div class="main">
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      <a class="anchor" id="Top"></a>
       <div class="block">
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         <p class="content-title">Protocols</p>
         <p class="content-title">Protocols</p>
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<a class="anchor" id="Parts"></a>
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        <h2>The Parts</h2>
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        <p>The 6 genes that were required to assemble various routes were obtained by PCR from plasmids kindly provided by Genomikon Inc. Each gene (AmpR, KanR, ClrR, GFP, RFP and aCP) exist as self contained cassettes that are flanked by BsaI sites. Parts were then generated according to the schematic shown below:</p>
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<img src="/wiki/images/c/c5/Making_parts_678x586.png" align="middle" style="width:100%;">
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         <p>Our project utilized the “Bead Assembly Method” developed through Project
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         <h2>Building the Plasmids</h2>
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          <a href="https://2009.igem.org/Team:Alberta">Genomikon</a> and Project
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        <p>Our plasmid assembly system relies upon the achievements of previous Team Alberta iGEM entries, 2009’s BioBytes and 2010’s Genomikon assembly methods. This process begins with origins of replication anchored at one end to magnetic beads in a suspension within a reaction microfuge tube. The anchored strands have a single, free-floating sticky end, onto which successive genes are ligated. Once the new gene has been ligated on, we use a magnet to hold the beads (along with the anchored DNA) inside the reaction tube, while washing away the rest of the reaction, including the enzyme, buffers, and any non-ligated DNA that remains. The beads are then resuspended in a new reaction mixture, containing the next ligation step.</p>
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          <a href="https://2010.igem.org/Team:Alberta/Tour/biobytes">Biobytes</a> by
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<img src="/wiki/images/b/b4/2013Alberta-Poster2.jpg" align="middle" style="width:100%;">
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          the 2010 and 2009 University of Alberta iGEM teams.</p>
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<p>Once all of the genes have been ligated, a tail-piece that complements the original bead-anchor DNA sequence is added, so that the finished product can be unbound from the beads and will close upon itself to form the circular plasmid. In this fashion, a four-gene, roughly 5000-base-pair plasmid is assembled in as little as an afternoon, cheaply and easily.</p>
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        <p>After creating a sample map (with a highly favorable first-place solution), the Bead Assembly protocol was 
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          modified to incorporate concentrations that were relevant to our project map. We call this our first working 
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          Four-node <a href="/wiki/images/9/9c/Obvious_TSP_protocol.pdf">Travelling Salesman Protocol</a>.</p>
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        <img src="/wiki/images/b/be/2013_Alberta_Bead_Assembly.png" style="width:100%;"></img>
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        <p>This assembly method allowed for sequential attachment of DNA pieces onto a magnetic bead or onto DNA pieces already anchored to the magnetic bead. This anchor allows for easy separation of the excess, unbound DNA during the wash step.</p>
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        <p>All constructs were initiated by ligating the origin of replication to the DNA attached to the beads (step 1) in microcentrifuge tubes. Once ligation was completed, the reaction medium was removed while the beads were kept in the tube using a magnet. A new reaction solution, containing the genes representing all possible paths from the origin city, was then added for ligation (step 2). Again, once ligation was completed the reaction medium was removed while the beads were kept in the tube. A new reaction solution, this time containing the linkers representing cities, was added for ligation (steps). These steps were repeated until the 10 steps necessary to builds our solutions (plasmids) were performed. <p>
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        <p>As can be seen from the DNA agarose gel figure, the efficiency of the sequential assembly reached 90%. The length of the constructs increased as expected with the addition of each new path and city (gene and linker). Few constructs were not incorporating the parts to be added in a step. This allowed us to obtain plasmids for all possible solutions in a single procedure using a few assembly steps (parallel production of multiple, diverse solutions).
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For more, see our step-by-step protocol of the <a href="/wiki/images/a/ae/2013_Alberta_Bead_assembly_protocol.pdf">Bead Assembly Method</a>.</p>
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Latest revision as of 03:46, 29 October 2013


Places

Top

The Parts

Building the Plasmids
Project Sections

Background

Overview

Results

Protocols

Submitted Parts

Accomplishments

Protocols

The Parts

The 6 genes that were required to assemble various routes were obtained by PCR from plasmids kindly provided by Genomikon Inc. Each gene (AmpR, KanR, ClrR, GFP, RFP and aCP) exist as self contained cassettes that are flanked by BsaI sites. Parts were then generated according to the schematic shown below:

Building the Plasmids

Our plasmid assembly system relies upon the achievements of previous Team Alberta iGEM entries, 2009’s BioBytes and 2010’s Genomikon assembly methods. This process begins with origins of replication anchored at one end to magnetic beads in a suspension within a reaction microfuge tube. The anchored strands have a single, free-floating sticky end, onto which successive genes are ligated. Once the new gene has been ligated on, we use a magnet to hold the beads (along with the anchored DNA) inside the reaction tube, while washing away the rest of the reaction, including the enzyme, buffers, and any non-ligated DNA that remains. The beads are then resuspended in a new reaction mixture, containing the next ligation step.

Once all of the genes have been ligated, a tail-piece that complements the original bead-anchor DNA sequence is added, so that the finished product can be unbound from the beads and will close upon itself to form the circular plasmid. In this fashion, a four-gene, roughly 5000-base-pair plasmid is assembled in as little as an afternoon, cheaply and easily.

Retrieved from "http://2013.igem.org/Team:Alberta/Protocols"