Biomod/2013/Todai/Result: Difference between revisions

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     <p class="paragraph">
     <p class="paragraph">
     TEM images confirm that our OCK has two domains. Comparing the observed structure to our design, one domain match the shape and size to plane-like domain. And the other domain matches to stick-like domain. Furthermore, in close watching the images, DNA well, which exists one side of plame-like domain, could be detected.
     TEM images confirm that our OCK has two domains. Comparing the observed structure to our design, one domain match the shape and size to plane-like domain. And the other domain matches to stick-like domain. Furthermore, in close watching the images, DNA well, which exists one side of plane-like domain, could be detected.
     </p>
     </p>
     <div class="res-conclusion">
     <div class="res-conclusion">
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         </p>
         </p>
     </div>
     </div>
   
      <div class="zairyou-heading">[Result & Discussion]</div>
       <div class="res-conclusion">
       <div class="res-conclusion">
       a) Optimum concentration of CuSO<sub>4</sub> to OCK
       a) Optimum concentration of CuSO<sub>4</sub> to OCK
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     </article>
     </article>
     <br>
     <br>
<!--Result&Discussion1-->
      <div class="zairyou-heading">[Result & Discussion]</div>
    <div class="res-conclusion">
    </div>
      <figure>
        <center>
        <img src="" width=640px height=360px>
            <figcaption> <b></b>
            </figcaption>
        </center>
      </figure>
    <p class="paragraph">
    </p>
    <div class="res-conclusion">
    </div>
   
   <br>
   <br>
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Revision as of 11:51, 26 October 2013

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<html> <head> <title>Result-Todai nanORFEVRE-</title> <style>

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<body>

<!--◆◆Result◆◆--> 

  <h1 class="big-title"><a name="Result">&nbsp;Result</a></h1>



  <h1 class="heading"><a name="Contents">&nbsp;Contents</a></h1>

<ul> <li><h3><a href="#STEP1">STEP 1: DNA strands assemble to form designed structures.</a></h3> <ul style="list-style: none;"> <li><a href="#Optimize_the_condition_to_assemble_OCK">1)Optimize the condition to assemble OCK</a></li> <li><a href="#Conformation_of_the_3D_structure_of_OCK_by_TEM">2)TEM imaging of the 3D structure of OCK</a></li></ul> </li> <li><h3><a href="#STEP2">STEP 2: Subunits stick in the membrane.</a></h3> <ul style="list-style: none;"> <li><a href="#Flotation_assay">1)Flotation asssay</a></li> <li><a href="#Preparation_of_GUVs">2)Preparation of GUVs</a></li> </ul> </li> <li><h3><a href="#STEP3">STEP 3: Recognition of target cells</a></h3> </li> <li><h3><a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a></h3> <ul style="list-style: none;"> <li><a href="#Oligomerization_by_streptavidin-biotin_complex">1)Oligomerization by streptavidin-biotin complex</a></li> <li><a href="#Tem_imaging_of_OCK_dimers_by_streptavidin-biotin_complex">2)TEM imaging of OCK dimers connected by streptavidin-biotin interaction</a></li> <li><a href="#Oligomerization_by_Click_reaction">3)Oligomerization by Click reaction</a></li> </ul> </li> 

  <h1 class="heading"><a name="Oligomeric_Cell_Killer_(OCK)">&nbsp;Oligomeric Cell Killer (OCK)</a></h1>

<!--◆◆STEP 1: DNA strands assemble to form designed structures.◆◆--> 

    <article>
     <h2 class="small-title"><a name="STEP1">&nbsp;STEP 1: DNA strands assemble to form designed structures.</a></h2>

<!--◆◆Optimize the condition to assemble OCK◆◆--> <h3><a name ="Optimize_the_condition_to_assemble_OCK"></a>1)Optimize the condition to assemble OCK</h3> <!--Method--> 

    <div class="zairyou-heading">[Method]</div>
      <figure class="figure-left">
        <img style="float:left; margin:0;margin-right:-10px;margin-bottom:10px;"
        src="http://openwetware.org/images/1/1e/OCKnaname-Todai.png" width="200px" height="200px" >
      </figure>

    <div id ="step0_1)">
      <p class="paragraph">
      The DNA nanostructure,
      <a target="_bramk" href="http://openwetware.org/wiki/Biomod/2013/Todai/Design#Oligomeric_Cell_Killer " style="color:#e00000">
      "Oligomeric Cell Killer"
      </a>
      , was designed to achieve our goal(--><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Project">Project</a>). 
      </p>

     <p class ="paragraph">

To know the optimum condition of the structure assembly, we did experiments in three conditions as follows. <ul style="position:relative;left:30px;"> <li>At different concentration of MgCl<sub>2</sub></li> <li>At different incubate temperature</li> <li>At different length of incubate time</li> 

       </ul>
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

<!--Result&Discussion1--> 

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

    <div class="res-conclusion">
   Optimum concentration of MgCl<sub>2</sub>
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/a/af/OOCK_Optimize_MgConc.png" width=640px height=360px>
           <figcaption> <b>Agarose-gel electrophoresis to research the optimum concentration of MgCl<sub>2</sub>

</b> 

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

    <p class="paragraph">

Fast-migrating species upon agarose-gel electrophoresis was yielded at 10~20mM MgCl<sub>2</sub> condition. At higher MgCl<sub>2</sub> concntration, a sub-band, which might be a dimer, appeared. 

    </p>
    <div class="res-conclusion">
    --->> Optimum concentration of MgCl<sub>2</sub>: 10mM   
    </div>

    <br>
    <br>

<!--Result&Discussion2--> 

    <div class="res-conclusion">
   Optimum incubate temparature
    </div>
  
    <figure>
       <center>
          <img src="http://openwetware.org/images/9/95/OOCK_Optimize_Temp-Todai.png" width=640px height=360px >
          <figcaption>
          <b>Agarose-gel electrophoresis to research the optimum temperature</b>
          </figcaption>
       </center>
    </figure>

   <p class="paragraph">

Fast-migrating species upon agarose-gel electrophoresis was yielded at 52.0 °C. 

    </p>
    <div class="res-conclusion">
    --->> Optimum temparature : 52.0 °C  
    </div>
    <br>
    <br>

<!--Result&Discussion2--> 

    <div class="res-conclusion">
    To decide optimum length of incubate time
    </div>

      <figure>
        <center>
          <img src="http://openwetware.org/images/9/94/OOCK_Optimize_Time-Todai.png" width=640px height=360px >
          <figcaption>
          <b>Agarose-gel electrophoresis to research the optimum time</b>
          </figcaption>
       </center>
      </figure>

   <p class="paragraph">

The band for 3h is fast migrated and sharp. 

    </p>

    <div class="res-conclusion">
    --->> Optimum incubate time : 3h 
    </div>

    <br>

<!--◆◆1.2 Conformation of the 3D structure of OCK by TEM◆◆--> 

    <h3><a name="Conformation_of_the_3D_structure_of_OCK_by_TEM"></a>2)Conformation of the 3D structure of OCK by TEM</h3>
    <!--Method-->
    
    <div class="zairyou-heading">[Method]</div>

    <div id ="step0_2)">
      <p class="paragraph">

Gel electro phoresis cannot visualize the 3D structure of OCK, so it was confirmed by Transmission electron microscopy (TEM) 

       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    </article>
    <br>

<!--Result&Discussion1--> 

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

    <div class="res-conclusion">
    TEM imaging of OCK
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/e/ea/Monomer-Todai.png" width=480px height=360px>
           <figcaption> <b>TEM image of OCK</b>
           Three monomers of OCK were observed in this figure. 
        
           </figcaption>
       </center>
      </figure>

    <p class="paragraph">
    TEM images confirm that our OCK has two domains. Comparing the observed structure to our design, one domain match the shape and size to plane-like domain. And the other domain matches to stick-like domain. Furthermore, in close watching the images, DNA well, which exists one side of plane-like domain, could be detected.
    </p>
    <div class="res-conclusion">
    </div>
    </article>

<!--◆◆2. Penetration◆◆--> <!--editting--> 

     <h2 class="small-title"><a name="STEP2"></a>&nbsp;STEP 2: Subunits stick in the membrane.</h2>
     <article>

<!--◆◆2.1 Flotation assay◆◆--> 

    <h3><a name=" Flotation_assay"></a>1) Flotation assay</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step3_1)">
      <p class="paragraph">
      OCK was designed to penetrate lipid bilayer. However, it is difficult to conclude the penetration of OCK. Therefore, we first did flotation assay to detect the interaction of OCK with lipid. 
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    <br>

<!--Result&Discussion1--> 

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

    <div class="res-conclusion">
    
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/5/5f/450pxOCK_Fluorescence-Todai.jpg" width=450px height=350px>
           <figcaption> <b>The fluorescence intensity of NIL (in liposome) in each fraction </b><br>
           The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3(lower layer).
           </figcaption>
       </center>
      </figure>
          <figure>
       <center>
        <img src="http://openwetware.org/images/f/fe/FlotationOCK12gel-Todai.png" width=480px height=360px>
           <figcaption> <b>1% Agarose gel electro phoresis of each fraction in sample 1, 2</b>
           </figcaption>
       </center>
      </figure>
          <figure>
       <center>
        <img src="http://openwetware.org/images/0/0d/FlotationOCK34gel-Todai.png" width=480px height=360px>
           <figcaption> <b>1% Agarose gel electro phoresis of each fraction in sample 3, 4</b>
           </figcaption>
       </center>
      </figure>
          <figure>
       <center>
        <img src="http://openwetware.org/images/f/ff/450px_FloatingOCKprofile-Todai.jpg" width=450px height=300px>

       </center>
      </figure>

    <p class="paragraph">
    With the condition of cholesterol +/ liposome+, the peak fraction was No.3, which coinced with peak fraction of liposome. In contrast, lacking of cholesterol or liposome, OCK exist mainly in fraction No.2.

As the peak fraction of OCK shifted from fraction No.2 to No.3, with the attachment of cholesterol and existence of liposome, we concluded that OCK stack in liposome. 

    </p>
    <div class="res-conclusion">
    --> OCK stack in liposome.
    </div>
    </article>

<!--◆◆2.2 Preparation of GUVs◆◆--> 

     <article>
    <h3><a name="Preparation_of_GUVs"></a>2) Preparation of GUVs</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step2_2)">
      <p class="paragraph">
     	GUV, Giant Unilamellar Vesicle, was prepared to visualize the sticking of OCK in membrane. The comparation between the fluorescence of OCK (Cy5) and GUV(NIL, Nile Red) was expected to suggest the sticking.
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    <br>

<!--Result&Discussion1--> 

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

    <div class="res-conclusion">
    
    </div>

          <figure>
       <center>
        <img src="http://openwetware.org/images/b/be/GUVconfocal_scale-Todai.png" width=450px height=350px>
           <figcaption> <b>The ratios of OCK in each fraction analyzed by the density of band.</b>
           </figcaption>
       </center>
      </figure>

    <p class="paragraph">

GUVs were observed with confocal laser scanning microscope (Carl Zeiss, LSM 5 Exciter). As the tracer of GUVs, 0.1 mol% Nile Red (Ex 553 nm, Em 637 nm) was used. 

About 10 um of GUVs were observed.

    </p>
    <div class="res-conclusion">
    --> OCK stack in liposome.
    </div>
    </article>

<!--◆◆STEP 3: Recognition of target cells.◆◆--> <!--◆◆STEP 4: The formed subunits oligomerize in solution.◆◆--> 

    <article>
     <h2 class="small-title"><a name="STEP4"></a>&nbsp;STEP 3: The formed subunits oligomerize in solution.</h2>

<!--◆◆4.1 Oligomerization by streptavidin-biotin complex◆◆--> 

    <h3><a name="Oligomerization_by_streptavidin-biotin_complex"></a>1) Oligomerization by streptavidin-biotin complex</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step3_1)">
      <p class="paragraph">
      Biotins are equipped to OCK for oligomerization. The experiment which confirmed that streptavidins induced oligomeriation. 
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    </article>
    <br>

<!--Result&Discussion1--> 

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

    <div class="res-conclusion">
    Streptavidins induced oligomerization
    </div>

      <figure>
       <center>
        <img src="http://openwetware.org/images/5/5b/Streptavidin_dimer-Todai.png" width=600px height=450px>

<figcaption>The mixing ratio of streptavidin to OCK was equal to 5:3, which means the mixing ratio of streptavidin to biotin was equal to 5:6 in the condition (L+R).</figcaption> 

       </center>
      </figure>
    </article>

<!--◆◆4.2 EMm imaging of dimers by streptavidin-biotin complex◆◆--> 

    <article>
         <h3><a name="Tem_imaging_of_OCK_dimers_by_streptavidin-biotin_complex"></a>2) TEM imaging of OCK dimers connected by streptavidin-biotin interaction</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step4_2)">
      <p class="paragraph">
      Dimers of OCKs were also imaged by TEM to confirm the bands observed in the experiment 3.1) originated from the dimers.
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   <br>
   <br>

    </article>
    <br>

<!--Result&Discussion1--> 

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

    <div class="res-conclusion">
    </div>
    <center>
     <table cellpadding="0" style ="position:relative;left:-30px;">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/c/c1/Dimerv2-Todai.png" width="300px" height="300px" >
       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/5/53/Dimer_2v2-Todai.png" width="300px" height="300px" >
       </figure>
       </td>
       </tr>
     </table>
     </center>
    <p class="paragraph">
    	Dimers of OCKs  were observed in this experiment and two of them were shown above. 
    </p>
    <div class="res-conclusion">
    -->The dimerization by streptavidin-biotin complex was confirmed.
    </div>

<!--◆◆4.3 Optimum concentration of CuSO4◆◆--> 

    <article>
    <h3><a name="Oligomerization_by_Click_reaction"></a>3) Oligomerization by Click reaction</h3>
    <!--Method-->
    <div class="zairyou-heading">[Method]</div>

    <div id ="step4_3)">
      <p class="paragraph">
      Azide and alkyne, which function as a reactive group of click reaction, are also equiped to OCK. It demands Cu<sup>+</sup> as catalyst, but too high concentration of Cu<sup>+</sup> (cation) might denaturate OCK like Mg<sup>2+</sup>. Therefore, we optimized the concentration of Cu<sup>+</sup> to OCK first, and then the optimum Cu<sup>+</sup> concentration to click reaction was investigated.
       (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
       protocols
       </a>
       )
       </p>
   </div>
   
     <div class="zairyou-heading">[Result & Discussion]</div>
      <div class="res-conclusion">
      a) Optimum concentration of CuSO<sub>4</sub> to OCK
    </div>
          <figure>
       <center>
        <img src="http://openwetware.org/images/5/5e/CuAlive-Todai.png" width=600px height=450px>

       </center>
      </figure>
             <div class="res-conclusion">
      -->Optimum concentration of CuSO<sub>4</sub>: 625 uM or less
    </div>
   <br>
   <br>
   </div>
      <div class="res-conclusion">
      b) Optimum concentration of CuSO<sub>4</sub> to click reaction
    </div>
          <figure>
       <center>
        <img src="http://openwetware.org/images/9/94/ClickResult-Todai.png" width=600px height=450px>

       </center>
      </figure>
      <p class ="paragraph">
      Product of click reaction appeared over 375 uM CuSO<sub>4</sub> concentration. Combining with the stability data, we decided to use 625 uM CuSO<sub>4</sub> condition.
             <div class="res-conclusion">
             --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
    </div>
    </article>
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