Difference between revisions of "Biomod/2013/Todai/Experiment"

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Revision as of 21:00, 18 September 2013

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       </div>
     </figure>
   </div>
   <br>

<div class="sidebar">

 <ul>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai">Home</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Project">Project</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Design">Design</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result">Result</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment">Experiment</a>
       <ul style="list-style-type: none;">

<li>

          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Contents">
          Contents</a></li>
          <li>
          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#PilotStudy">
          Pilot Study</a></li>
          <li>
          <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols">
          Protocols</a></li>
       </ul>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Team">Team</a>
    </li>
    <li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Sponsors">Sponsors</a>
    </li>
 </ul>

</div>


<div class="Return-Logo">

 <a href="#TOP">
   <figure>
     <img src="http://openwetware.org/images/b/b1/Return-top-0828new.png" width:60px height:60px>
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<html> <head> <title>Experiment-Todai nanORFEVRE-</title> <style>

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<body>

<!--Experiment-->

   <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>
   <div id="Explist">
   <ul >
      <li><div class="mokuji"><a href="#Contents">Contents</a></div></li>
      <li><div class="mokuji"><a href="#PilotStudy">Pilot Study</a></div></li>
      <li><div class="mokuji"><a href="#Protocols">Protocols</a></div></li>
   </ul>
   </div>
   <br>

<!--Contents-->

  <article>
    <h1 class="title"><a name="Contents">&nbsp;Contents</a></h1>
      <article>
        <ul class="Contents-list">
        <li>
        <a name="#Assembling_of_DNA_structure">
        <b>Assembly of DNA nanostructure</b>
        </a>
        -->(See <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result" style="color:#E00000">Result</a>)
        <br>
        Optimize the assembly condition of the DNA nanostructure,"Cylinder in barrel"
        </li>
        <br>


        <li><a name="#flotation_assay"><b>Flotation assay</b></a>
        <ul class="Contents-list">
          <li><a href ="#Preparation_of_liposome">Preparation of liposome</a>
          <br>
          Making of liposome for floatation assay
        </li>
    
        <li>
        <a href ="#Flotation_assay_of_liposome_and_DNA_origami">
        Floatation assay of liposome and DNA origami
        </a>

<br>

        Assay to check the penetration of DNA origami

<br> </li>

        </ul>
        <br>
        <li>
        <a href ="#Comparision_of_dimerization">
        <b>Comparision of dimerization method</b>
        </a>
        <ul class="Contents-list">
          <li>
          <a href ="#Click_reaction_via_(3+2)_cycloaddition">
          Click reaction via (3+2) cycloaddition
          </a>
          <br>
          Optimize the reaction condition for click chemistry 
          <br>
          </li>
          <li><a href ="#Azobenzene">Azobenzene</a>
          <ul class="Contents-list">
            <li><a href ="#Synthesis_of_Tube(Research_for_azobenzene)">Synthesis of tube</a>
            <br>
            Optimize the assembly condition pf the DNA origami tube to be equipped with azobenzene
            <br>
            </li>
            <li><a href ="#Synthesis_of_Motif(Research_for_azobenzene)">Synthesis of tube</a>
            <br>
            Optimize the assembly condition of the DNA motif to be equipped with azobenzene
            </li>
            <br>
          </ul>
          </li>
     </ul>
     </li>
     </ul>
 </article>
</article>


<!--Pilot Study-->

  <article>
  
    <h1 class="title"><a name="PilotStudy">&nbsp;Pilot Study</a></h1>
    

<!--Liposome making-->

  <article>
  
  <h2 class="PS_title"><a name="Assembling_of_DNA_structure">&nbsp;1.Assembly of DNA nanostructure</a></h2>
  <p class="paragraph">
  The assembly of DNA structure is explained in the
  <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result" style="color:#e00000">Result page</a>.
  <br>   
  <br>
  <h2 class="PS_title"><a name="flotation_assay">&nbsp;2.Flotation assay</a></h2>
  <div class="mini-title">
      <a name="Preparation_of_liposome">2-1. Preparation of liposome</a>
  </div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/b/b5/640px_liposomeDLS-Todai.png" width=640px height=360px >
<figcaption> <b>The result of DLS </b>
     </figcaption>
       </center>
      </figure>

<div class="zairyou-heading">[Discussion]</div>

    <p class="paragraph">In flotation assay, uniformly-sized liposomes are required because liposomes should have 

same buoyancy. Moreover, liposomes must have enough radiuses (about 100nm in radius) to float in sucrose buffer.

Using Extruder device(Avanti), we prepared liposome of 120nm in radius.

    </p>
  </article>
  <br>

<!--Flotation assay-->

  <article>
  <div class="mini-title">
      <a name="Flotation_assay_of_liposome_and_DNA_origami">2-2. Floatation assay of liposome and DNA origami</a>
      <figure>
       <center>
        <img src="http://openwetware.org/images/1/1d/640pxflotationassay-Todai.jpg" width=300px height=300px >
<figcaption> <b>Result of agarose gel electrophoresis of the sample of flotation assay</b> <br>
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is 

integrated into DNA origami(Rect tile<sup>[1]</sup>) ,is observed. Fraction1 is the liquid in the top layer, and fraction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also loaded on the extreme right lane. </figcaption>

       </center>
      </figure>
      <figure>
       <center>
        <img src="http://openwetware.org/images/0/0d/300pxNILGraph-Todai.PNG" width=350px height=350px >
<figcaption> <b>Fluorescence intensity of the samples of flotation assay(DNA Rect tile +liposome)</b><br>

Although the size of liposome might change during the flotation assay(data not shown), the intensity of the fluorescence of NIL(Nile Red, ex 500nm, em 550~700nm ) suggests the amount of lipid membrane,liposome. The fluorescence spectrum of water was subtracted as background

     </figcaption>
       </center>
      </figure>

<br>

  <div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph">

To confirm the flotation assay, mixed tiles(DNA origami) and liposomes were assayed. Five samples (fraction 1,2,...,5, from the top) were retrieved from supermetant liquid and a sample(fraction 6) from precipitation by the addition of buffer used in assay. When the sample, tile mixed with liposomes, were assayed, tiles were observed in the top layer. The distribution of liposomes is observed by the fluorescence of NIL(Nile Red).

    </p>
  <br>

</article>


<!--Click Reaction-->

  <h2 class="PS_title"><a name="Comparision_of_dimerization">3. Comparision of dimerization method</a></h2>
  <article>
  <div class="mini-title">
      <a name="Click_reaction_via_(3+2)_cycloaddition">
      3-1. Click reaction via (3+2) cycloaddition<sup>[4]</sup>
      </a>
  </div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/a/ab/450pxclick0828-Todai.jpg" width=480px height=360px >
<figcaption> <b>Result of urea gel electrophoresis of the sample of click reaction</b><br>
     </figcaption>
       </center>
      </figure>

<div class="zairyou-heading">[Discussion]</div>

    <p class="paragraph">Copper(Ⅰ) catalyzed click reaction was used to dimerize of oligo DNA(length of 20bp and 14bp). The time cause of the reaction indicate that the click reaction is so quick(<5min).


    </p>
  <br>

<!--Synthesis of tube (Research for azobenzene)-->

  <article>
     <div class="mini-title">
     <a name ="Azobenzene">3-2. Azobenzene</a>
    </div>
    <div class="mini-title">
     <a name="Synthesis_of_Tube(Research_for_azobenzene)">&nbsp;&nbsp;3-2-1. Synthesis of tube<sup>[2],[3]</sup>(Research for azobenzene)</a>
    </div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/b/b2/640x360px_tube_result-Todai.png" width=640px height=360px >
        <figcaption>
        <b>results of the electrophoresis of DNA-tube</b><br>
        </figcaption>
       </center>
      </figure>
    <div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph"> 
    We examined how DNA-tube was synthesized efficiently by using the method which is introduced in “Rapid Folding of 

DNA into Nanoscale Shapes at Constant Temperature” (Jean-Philippe J. Sobczak et al, Science, 2012, 338, 1458)

   <sup>[2]</sup>. In the figure above, two bands derived from scaffold or DNA-tube were showed with cursors. At 56.4℃, the scaffold 

band was diminished. In contrast, the DNA-tube band was concentrated. In fact, the ratio of DNA- tube band to scaffold band was the greatest at this temperature, which means we succeeded in synthesizing DNA- tube efficiently and improving the yield of DNA-tube.

    </p>
  <br>


<!--Synthesis of Motif (Research for azobenzene)-->

  <article>
  <div class="mini-title">
      <a name="Synthesis_of_Motif(Research_for_azobenzene)">&nbsp;&nbsp;3-2-2. Synthesis of Motif<sup>[5]</sup></a>
  </div>
      <figure>
       <center>
        <img src="http://openwetware.org/images/a/a8/480px_electrophoresis_of_T-motif_improved-Todai.png" width=480px height=360px >
        <figcaption>
         <b>Result of agarose gel electrophoresis of T-motif (wheel)</b>
         <br>
         The result of 1.5% agarose gel electrophoresis(100V) for 33min. 
        </figcaption>
       </center>
      </figure>
    <div class="zairyou-heading">[Discussion]</div>
    <p class="paragraph"> 
    In this measurement, it is difficult to distinguish between final structure band and monomer band.
    Next time, Native PAGE will be used instead of agarose gel. 
    </p>
  <br>


<!--Protocols-->

  <article>
    <h1 class="title"><a name="Protocols">&nbsp;Protocols</a></h1>

<!--scaffold+staple+cholesterol-->

  <article>
  <div class="mini-title">
      <a name="Assembling_of_DNA structure (scaffold+staple+cholesterol)">1.Assembly of DNA structure (scaffold+staple)<sup>[2]</sup></a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>M13mp18ss (0.1μM)</th>
   <td>5μL</td>
   </tr>
   <tr>
   <th>staple mix (0.68μM)</th>
   <td>4μL</td>
   </tr>
   <tr>
   <th>10x buffer</th>
   <td>1μL</td>
   </tr>
   </table>
   <br>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]<sup>[2]</sup></div>
   <ul class="procedure-list">
     <li>5μL of M13mp18ss, 4μL of staple mix and 1μL of 10×buffer were mixed, pipetting.</li>
     <li>The solution was kept at 45 degree C for 4h.</li>
   </ul>
   </article>
   <br>

<!--liposome-->

  <article>
  <h2>2.Flotation assay</h2>
  <div class="mini-title">
      <a name="Preparation_of_liposome">2-1. Preparation of liposome</a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>150mM aqueous KCl solution</th>
   <td>3mL</td>
   </tr>
   <tr>
   <th>POPC</th>
   <td>3mg</td>
   </tr>
   <tr>
   <th>Chloroform (99.0%)</th>
   <td>3mL</td>
   </tr>
   <tr>
   <th>40μM Nile Red solution</th>
   <td>0.1mL</td>
   </tr>
   </table>
   <br>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>POPC were dissolved in 3mL of Chloroform.</li>
     <li>A lipid film was formed by evaporating 3mL of POPC solution in a 50mL eggplant flask, using a rotational 

evaporator for 5 minutes.</li>

     <li>The flask was kept under vacuum overnight to evaporate remaining chloroform.</li>
     <li>The lipid film was resuspended in 3mL of a 150mM aqueous KCl solution.</li>
     <li>The solution was filtered through 200nm polar filter with extruder to even the size of liposome.</li>
     <li>The size of liposome was measured with DLS (Viscotek 802 DLS).</li>
     <li>The solution was kept at 3 degree C until usage.</li>
   </ul>
   </article>
   <br>

<!--flotation assay-->


  <article>
  <div class="mini-title">
      <a name="Flotation_assay_of_liposome">2-2. Flotation assay of liposome and DNA origami</a>
  </div>


<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
 <div class="zairyou-heading">・Sucrosebuffer</div>
   <div class="zairyou-heading-sub">a) 0.375M Sucrose buffer</div>
   <table>
   <tr>
   <th>HEPES-KOH(pH7.6)</th>
   <td>50mM</td>
   </tr>
   <tr>
   <th>KCl</th>
   <td>100mM</td>
   </tr>
   <tr>
   <th>MgCl<sub>2</sub></th>
   <td>10mM</td>
   </tr>
   <tr>
   <th>Sucrose</th>
   <td>0.375M</td>
   </tr>
   </table>
   <div class="zairyou-heading-sub">b) 1.25M Sucrose buffer</div>
   <table>
   <tr>
   <th>HEPES-KOH(pH7.6)</th>
   <td>50mM</td>
   </tr>
   <tr>
   <th>KCl</th>
   <td>100mM</td>
   </tr>
   <tr>
   <th>MgCl<sub>2</sub></th>
   <td>10mM</td>
   </tr>
   <tr>
   <th>Sucrose</th>
   <td>1.25M</td>
   </tr>
   </table>
   <div class="zairyou-heading-sub">c) 1.6M Sucrose buffer</div>
   <table>
   <tr>
   <th>HEPES-KOH(pH7.6)</th>
   <td>50mM</td>
   </tr>
   <tr>
   <th>KCl</th>
   <td>100mM</td>
   </tr>
   <tr>
   <th>MgCl<sub>2</sub></th>
   <td>10mM</td>
   </tr>
   <tr>
   <th>Sucrose</th>
   <td>1.6M</td>
   </tr>
   </table>
   <br>
   <div class="zairyou-heading-sub">・liposome </div>
   <div style="font-size:110%;position:relative;left:60px;">
   1mg/ml( -->
   <a href="#Preparation_of_liposome" style="color:#e00000">Preparation of liposome</a>)
   </div>
   <div class="zairyou-heading-sub">・DNA origami(Rect tile<sup>[1]</sup>) </div>
    <div style="font-size:110%;position:relative;left:60px;">~30nM</div>
   <br>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]</div>
   <ul class="procedure-list">
     <li>sample + sucrose buffer were layered in this order on centrifuge tubes.</li>
     <li>The tubes were centrifugalized.</li>
     <li>Samples were fructionized.</li>
     <li>Precipitation was resuspended with sucrose buffer.</li>
     <li>Samples were measured by the fluorescence of NIL(ex 500nm, em 550nm~700nm) and agarose gel electrophoresis.</li>
   </ul>
   </article>
   <br>


<!--Click Reaction-->

  <article>
  <h2>3. Comparision of dimerization method</h2>
  <div class="mini-title">
      <a name="Click_reaction_via_(3+2)_cycloaddition">
      3-1. Click reaction via (3+2) cycloaddition
      </a>
  </div>

<!--Reagent-->

     <div class="zairyou-heading">[Reagent]</div>
     <br>
   <table>
   <tr>
   <th>azide solution (10μM)</th>
   <td>3μL</td>
   </tr>
   <tr>
   <th>alkyne solution (10μM)</th>
   <td>3μL</td>
   </tr>
   <tr>
   <th>CuSO4 solution (50mM)</th>
   <td>1μL</td>
   </tr>
   <tr>
   <th>THTA solution (100mM)</th>
   <td>1μL</td>
   </tr>
   <tr>
   <th>sodium ascorbate solution (100mM)</th>
   <td>1μL</td>
   </tr>
   </table>
   <br>

<!--Procedure-->

  <div class="zairyou-heading">[Procedure]<sup>[4]</sup></div>
   <ul class="procedure-list">
     <li>The above all solutions were mixed, using a vortex.</li>
     <li>The solution was kept at room temperature.</li>
   </ul>
   </article>
   <br>

<!--Research for azobenzene-->

  <article>
  <div class="mini-title">
      <a name="Research_for_azobenzene)">
      3-2. Research for azobenzene
      </a>
  </div>


<!--Procedure-->

  <div class="zairyou-heading">[Procedure]<sup>[2],[3],[5]</sup></div>
   <p class="paragraph">
   The procedure of synthesis of tubes and motifs were refered to previous researches([2],[3],[5]) 
   </p>
   </article>
   <br>
   <br>


<!--Reference-->

    <h1 class="title"><a name="Reference">&nbsp;Reference</a></h1>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [1] Folding DNA to create nanoscale shapes and patterns
       </a>
       </div>
          <div class="reference-author">
          Rothemund, P. W.
          </div>
             <div class="reference-journal">
             Nature 440, 297–302 (2006)
             </div>
    </div>
    <div>
       <div class="reference-title">
       <a name="proref-1">
       [2] Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature
       </a>
       </div>
          <div class="reference-author">
           Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz
          </div>
             <div class="reference-journal">
             Science, 2012, 338, 1458
             </div>
    </div>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Nanostructure
       </a>
       </div>
          <div class="reference-author">
          Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
          </div>
             <div class="reference-journal">
             Journal of the American Chemical Society, 2012, 134, 2852-2855
             </div>
    </div>
    <div>     
       <div class="reference-title">
       <a name="proref-1">
       [4] the protocol of Jena Bioscience GmbH
       </a>
       </div>
         <div class="reference-journal">
          <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
          http://www.jenabioscience.com</a>
         </div>
    </div>
    <div>
       <div class="reference-title">
       <a name="proref-1">
       [5] Substrate-Assisted Assembly of Interconnected Single-Duplex DNA Nanostructures
       </a>
       </div>
          <div class="reference-author">
          Shogo Hamada, Satoshi Murata Prof.
          </div>
             <div class="reference-journal">
             Angewandte Chemie International Edition,2009,48(37),6820–6823
             </div>
    </div>
             
 </article>
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