Team:UC Davis/Notebook/Protocols

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    <ul>
    <ul>
  <li style='color:#014457; cursor:default'><a>teams</a></li>
  <li style='color:#014457; cursor:default'><a>teams</a></li>
-
                    <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis">Page</a></li>
+
                    <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols">Page</a></li>
                 <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis/Notebook/Protocols&amp;action=edit&amp;redlink=1">Discussion</a></li>
                 <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis/Notebook/Protocols&amp;action=edit&amp;redlink=1">Discussion</a></li>
               <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&action=edit">Edit</a></li>
               <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&action=edit">Edit</a></li>
-
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=history">History</a></li>
+
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&amp;action=history">History</a></li>
-
                 <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis">Move</a></li>
+
                 <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis/Notebook/Protocols">Move</a></li>
-
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=watch">Watch</a></li>
+
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&amp;action=watch">Watch</a></li>
  <li><a href="https://igem.org/Login">Log in</a></li>
  <li><a href="https://igem.org/Login">Log in</a></li>
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   <div id="newnavi">
   <div id="newnavi">
     <ul class="newmenu">
     <ul class="newmenu">
-
        <li ><a href="https://2012.igem.org/" title="Back to iGEM">iGEM</a></li>
+
    <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
 +
          <ul>
 +
          <li><a target="new" href="https://2012.igem.org/">Main iGEM</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li>
 +
          </ul>
 +
        </li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
-
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
+
         <li ><a title="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
           <ul>
           <ul>
-
             <li ><a href="./Data.htm ">Data 1</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity" title="Data">Cutinase Activity</a></li>
-
             <li ><a href="./Data.htm ">Data 2</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
-
            <li ><a href="./Data.htm ">Data 3</a></li>
+
title="Data">Ethylene Glycol</a></li>
-
        </ul>
+
<li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Modeling"
 +
title="Data">Modeling</a></li>
 +
 
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li>
 +
          </ul>
         </li>
         </li>
-
         <li class="selected"><a title="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
+
         <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
           <ul>
           <ul>
-
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook_Overview ">Notebook Overview</a></li>
 
-
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a></li>
 
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a></li>
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a></li>
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a></li>
           </ul>
           </ul>
         </li>
         </li>
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         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
-
         <li ><a title="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
+
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
           <ul>
           <ul>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
-
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Modular Engineering</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Module Engineering</a></li>
-
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
 +
            <li ><a title="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering </a>
 +
  <ul>
 +
                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Background</a></li>
 +
        <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution">Directed Evolution</a></li>
 +
                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain">Rational Engineering </a></li>
 +
</ul>
 +
</li>
           </ul>
           </ul>
         </li>
         </li>
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<div id="myleftrightbox">
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<a href="https://2012.igem.org/Team:UC_Davis/Notebook"><img src="https://static.igem.org/mediawiki/2012/2/24/UCD_notebook_small_banner.jpg"></a>
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<a href="https://2012.igem.org/Team:UC_Davis/Project/Notebook"><img src="https://static.igem.org/mediawiki/2012/2/24/UCD_notebook_small_banner.jpg"></a>
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<a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols"><img src="https://static.igem.org/mediawiki/2012/0/03/UCD_protocols_small_banner.jpg"></a>
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<a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery"><img src="https://static.igem.org/mediawiki/2012/4/43/UCD_Gallery_small_banner-1.jpg"></a>
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<center>
<center>
-
<a href="http://www.fishersci.com" target="_blank"><img src="https://static.igem.org/mediawiki/2011/a/a4/UCD_Fisher_Logo.gif" width="200"></a>
+
<a href="http://www.cs.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/6/6b/UCD_Computer_sponsor.jpg" width="200"></a>
</center>
</center>
<center>
<center>
<a href="http://www.bme.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/4/40/UCD_BME_logo_minimal_copy.png" width="200 height="70"></a>
<a href="http://www.bme.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/4/40/UCD_BME_logo_minimal_copy.png" width="200 height="70"></a>
 +
</center>
 +
 +
<center>
 +
<a href="http://www.fishersci.com" target="_blank"><img src="https://static.igem.org/mediawiki/2011/a/a4/UCD_Fisher_Logo.gif" width="200"></a>
 +
</center>
 +
 +
<center>
 +
<a href="http://www.arcadiabio.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/4/46/UCD_Arcadia_sponsor.jpg
 +
" width="200"></a>
 +
</center>
 +
 +
<center>
 +
<a href="http://provost.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/8/82/UCD_Provost_sponsor.jpg
 +
" width="200"></a>
 +
</center>
 +
 +
<center>
 +
<a href="http://www.research.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/9/99/UCD_Research_sponsor.jpg" width="200"></a>
 +
</center>
 +
 +
<center>
 +
<a href="http://ucomm.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/b/b4/UCD_Communications_sponsor.jpg" width="200"></a>
 +
</center>
 +
 +
<center>
 +
<a title="" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/25/UCD_Schultz_sponsor.jpg
 +
" width="200"></a>
</center>
</center>
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<ul>
<ul>
<li>Mix reactants, adding enzyme last.
<li>Mix reactants, adding enzyme last.
-
<li>Place at 37 C for 3-5 hours.
+
<li>Place at 37 °C for 3-5 hours.
-
<li>If not purifying or running on a gel immediately, increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
+
<li>If not purifying or running on a gel immediately, increase to 80 °C for 20 minutes to kill enzymes (some enzymes need only a 65 °C heatkill, check enzyme).
<li>Run on a gel and extract product.
<li>Run on a gel and extract product.
</ul>
</ul>
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     </div>  
     </div>  
-
   <p class="menu_head"> EMS (Ethyl Methane Sulfonate Mutagenesis)</p>
+
   <p class="menu_head"> EMS (Ethyl methanesulfonate Mutagenesis)</p>
     <div class="menu_body">
     <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
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<ul>
<ul>
<li>EMS (Sigma)</li>
<li>EMS (Sigma)</li>
-
<li>50mL Conical Corning tubes</li>
+
<li>50mL conical Corning tubes</li>
<li>15mL Falcon tubes</li>
<li>15mL Falcon tubes</li>
<li>LB Broth</li>
<li>LB Broth</li>
<li>Ethylene Glycol Agar Plates</li>
<li>Ethylene Glycol Agar Plates</li>
-
<li>Serological pipets (10mL) for cell re-suspension</li>
 
-
<ul>
 
-
<li>PipetAide</li>
 
-
</ul>
 
</ul>
</ul>
<p>Procedure</p>
<p>Procedure</p>
<ul>
<ul>
-
<li>Prepare a 10mL liquid culture in LB in a 50mL Conical tube and grow it overnight until it reaches OD~0.2.</li>
+
<li>1. Prepare a 10mL liquid culture in LB in a 50mL conical tube and grow it overnight until it reaches OD 0.2.</li>
<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
<li>Wash twice with 10mL Buffer A.</li>
<li>Wash twice with 10mL Buffer A.</li>
-
<li>Pipet up and down to re-suspend pellet</li>
+
            <ul>
-
<li>Pellet cells as in step 2</li>
+
                <li>Pipet up and down to mix, pellet cells, decant supernatant.</li>
-
<li>Decant supernatant in waste</li>
+
            </ul>
-
<li>Bleach waste </li>
+
<li>Re-suspend the pellet in 5mL of Buffer A and transfer the medium to a Falcon tube.</li>
 +
<li>Add (35μL, 70μL, or 105μL) of EMS into each tube of re-suspended cells. </li>
 +
<li>Close tubes and parafilm the lid 2X.</li>
 +
<li>Vortex to mix and place in a secondary container (50mL conical tube).</li>
 +
<li>Transfer to mixing platform and agitate at ~30 rpm at 37°C.</li>
 +
<li>Withdraw samples at fixed time points.</li>
 +
<li>At each time point, take 1mL aliquots of the sample and place in a 15mL Falcon tube. Place in a secondary container (50mL Conical tube) and spin at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 5 minutes. </li>
 +
<li>Discard supernatant in waste container. </li>
 +
<li>Wash twice in 5mL of Buffer A and discard supernatant in waste container.</li>
 +
<li>Re-suspend in 5mL of Buffer A and titer for viable cells.</li>
 +
<li>Add 0.5mL of mutagenized culture into 10mL of LB broth in a 50mL conical tube.</li>
 +
<li>Grow cultures overnight at 37°C and plate for mutants on EG agar plates.</li>
 +
<li>Look for viable cells</li>
 +
</ul>
 +
<p>Safety: Health Hazards</p>
 +
<ul>
 +
<li>Acute toxicity (oral, dermal, inhalation), category 4 </li>
 +
<li>Skin irritation, category 2 </li>
 +
<li>Eye irritation, category 2 </li>
 +
<li>Skin sensitization, category 1 </li>
 +
<li>Specific Target Organ Toxicity – Single exposure, category 3</li>
 +
<li>LD50 – 470mg/kg</li>
</ul>
</ul>
 +
 +
<p>Recommended Safety & Handling</p>
 +
<ul><li>Always work in fume hood and wear lab coat, goggles, and gloves.</li></ul>
 +
     </div>
     </div>
 +
<p class="menu_head">Construction of a Transposase Illumina Sequencing Library</p>
 +
    <div class="menu_body">
 +
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Extract genomic DNA using any proprietary gDNA extraction kit. Once DNA is extracted, quantify your DNA and run it on an agarose gel to check DNA quality.</li>
 +
<li>Prepare Illumina Adapters for Transposase Priming, Mix the Following:
 +
      <ul><li>10µM No_PCR_Adapter_1: 10µL</li>
 +
      <li>10µM No_PCR_Adapter_2: 10µL</li>
 +
      <li>10µM Mosaic Element: 20µL</li>
 +
      <li>5M NaCl: 1.2µL</li>
 +
      <li>Incubate at 98C for 5 mins and let cool to room temperature</li></ul></li>
 +
<li>Prime Transposase
 +
      <ul><li>Transposase: 10µL</li>
 +
      <li>Annealed adapter (above): 5µL</li>
 +
      <li>Water: 15µL</li>
 +
      <li>Incubate at room temperature for 20-30 mins.</li></ul></li>
 +
<li>Tagmentation Reaction
 +
      <ul><li>Primed Transposase: 10µL (depends on the concentration of available Tn5)</li>
 +
      <li>200ng of gDNA extration: XµL</li>
 +
      <li>5x Tn5 Buffer: 3µL</li>
 +
      <li>Water: up to 15µL</li>
 +
      <li>Run multiple reactions in parallel to achieve appropriate amount of input DNA for sequencing platform</li>
 +
      <li>Incubate at 55C for 10 mins, and immediately pool reactions and clean up using a PCR clean up kit, elute with 15µL water</li></ul></li>
 +
<li>Gap Filling
 +
      <ul><li>Run a typical PCR reaction, without oligos, at 72C for 5 mins</li></ul></li>
 +
<li>Size Selection
 +
      <ul><li>Run gap filling products on 2% agarose gel, size select for the appropriate size required for your sequencing platform</li></ul></li>
 +
<li>Quality check your library
 +
      <ul><li>Run bioanalyzer to check your library quality, and qPCR to quantify your library before submission.</li></ul></li>
 +
<li>SUBMIT!</li>
 +
</ul>
</div>
</div>
 +
 +
<p class="menu_head">Cutinase Expression and Western Blot</p>
 +
    <div class="menu_body">
 +
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Prepare a starter culture of MG1655 <i>E. coli</i> with pBad regulated and his-tagged LC-Cutinase along with a negative control.</li>
 +
<li>Inoculate new 5 mL cultures from the starter cultures at an OD 600 of about 0.05.</li>
 +
<li>Let the cultures grow until and OD 600 of 0.5, then inoculate two 100 mL cultures (one for the his-tagged protein and one for the negative control). </li>
 +
<li>Once these cultures are at an OD of 0.8, separate into two 50 mL cultures. Induce one at 10 uM, leave the other uninduced.</li>
 +
<li>Take 1 mL samples at different time points.</li>
 +
<li>Centrifuge samples at 5000g for 5 minutes then take off and save media.</li>
 +
<li>Wash cells with water.</li>
 +
<li>Resuspend with 300 uL of B-PER Protein Extraction Reagent and follow the instructions provided with that kit. After this, there should be samples of culture media, and soluble and insoluble whole cell lysate.</li>
 +
<li> Take 15 uL of each sample, including controls, and run on western blot.</li>
 +
 +
    </div>
 +
 +
<p class="menu_head">pNPB Assay</p>
 +
    <div class="menu_body">
 +
 +
<p>Materials</p>
 +
<ul>
 +
<li>pNPB buffer (should be made and used in fumehood due to acetonitriles toxicity)
 +
<ul><li>10mM pNPB in acetonitrile</li>
 +
<li>1:4:95 ratio of acetonitrile pNPB solution, 100% ethanol and 50mM Tris-HCL (pH 8)</li></ul></li>
 +
<li>LB with specific resistance</li>
 +
<li>Cell Culture</li>
 +
<li>96 well plate</li></ul>
 +
<br>
 +
<p>Protocol</p>
 +
<ul><li>First assign each well of the plate except for outer wells which should be filled with LB to prevent evaporation of medium.</li>
 +
<li>Fill wells with 95 uL of LB</li>
 +
<li>Fill wells with 5 uL of cell culture</li>
 +
<li>In fumehood, add 100 uL of bufer to each well</li>
 +
<li>Run in Tecan taking both ODs and absorbance 405 readings</li>
 +
 +
    </div>
 +
 +
<p class="menu_head">SDM (Site Directed Mutagenesis)</p>
 +
    <div class="menu_body">
 +
 +
<p>Materials</p>
 +
<ul>
 +
<li>Pfu turbo</li>
 +
<li>10X Pfu turbo buffer</li>
 +
<li>dNTPs (10mM)</li>
 +
<li>Forward and reverse primers (0.1ug/uL, see methods section for design tips)</li>
 +
<li>dH<sub>2</sub>0</li>
 +
<li>Dpn1</li>
 +
<li>Competent cells</li>
 +
</ul>
 +
<br>
 +
<p>Methods</p>
 +
<ul>
 +
<li>Primer Design</li>
 +
<ul>
 +
<li>Forward primer should be between 25 and 45 bases in length and contain the desired mutation in the center with correct sequences on both sides; the reverse primer is the reverse complement of this.</li>
 +
<li>Primers should have a minimum GC content of 40% and terminate in one or more C's or G's.</li>
 +
<li>Tm should be greater or equal to 78°C and can be calculated as follows:</li>
 +
<li>Tm = 81.5 + 0.41(%GC) - 675/(length in bases) - %mismatch</li>
 +
</ul></ul>
 +
<ul>
 +
<li>Reaction</li>
 +
<ul>
 +
<li>Template DNA: 1 µL</li>
 +
<li>10X Buffer: 5 µL</li>
 +
<li>Forward Primer (100 ng/µL): 1µL</li>
 +
<li>Reverse Primer (100 ng/µL): 1µL</li>
 +
<li>10mM dNTPs: 1µL</li>
 +
<li>Pfu Turbo (Stratagene): 1µL</li>
 +
<li>MilliQ H<sub>2</sub>0: 40µL</li>
 +
</ul>
 +
<li>PCR Program</li>
 +
<ul>
 +
<li>95°C for 1 minute</li>
 +
<li>95°C for 50 seconds, 60°C for 50 seconds, 68°C for 1 minute/kb of plasmid length -- repeat this step 17 times, or 18 cycles total</li>
 +
<li>68°C for 7 minutes</li>
 +
<li>4°C hold</li>
 +
<li>Following PCR - save 4µL of PCR reaction.</li>
 +
<li>To remaining 46µLs add 1µL of Dpn1 to PCR reaction (PCR cleanup is not necessary). Incubate at 37°C for 1-2 hours to digest parental DNA. Run 5µL of the digested reaction on a gel and compare to the undigested parental plasmid - there should be some difference in band pattern. Transform into competent cells (PCR cleanup is not necessary).</li>
 +
</ul>
 +
 +
<br>
 +
<li>Protocol Adapted From <a href="http://mcmanuslab.ucsf.edu/protocol/site-directed-mutagenesis-stratagene-protocol">Here</a>
 +
</ul>
 +
    </div>
 +
 +
 +
 +
 +
</div>
 +
 +
 +
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-
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+
<li style="float:left ;margin:0 10px;"><a
-
list-style-type:none;color:#000000"><li>Data One </li><li>Data Two </li><li>Data Three </li></ul></a> </li>
+
title="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p></a> <ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
"><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity ">
 +
Cutinase Activity</a> </li><li><a style="color:#000000 "
 +
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 +
Ethylene Glycol</a> </li><li><a style="color:#000000 "
 +
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 +
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-
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+
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-
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+
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 +
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 +
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 +
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 +
"><li><a style="color:#000000 " href="https://2012.igem.org/Main_Page
 +
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 +
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 +
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 +
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