Biomod/2014/Kashiwa/Highlights

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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa"><img src="http://openwetware.org/images/e/ec/LogoKashiwa.png" onmouseover="this.src='http://openwetware.org/images/7/7a/Logo2Kashiwa.png'" onclick="this.src='http://openwetware.org/images/1/1d/Logo2.5.png'" onmouseout="this.src='http://openwetware.org/images/e/ec/LogoKashiwa.png'" height="80px" width="120px" name="def"></a>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROJECT</span></a>
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       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Background</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Motivation</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#3" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Project Goals</span></a></li>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EARLY TRIAL&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Design</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Approaches</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">DESIGN</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li>
     </ul>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EXPERIMENT&nbsp;&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Highlights</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Receptor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Motor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;&nbsp;DISCUSSION&nbsp;</span></a>
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       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Achievements</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Future</span></a></li>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Protocols" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROTOCOL</span></a>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">TEAM</span></a>
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       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Members</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Sponsors</span></a></li>
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</div> <br> <br> <br> <div class="page-back"><a href="#top"> <img src="http://openwetware.org/images/2/2a/Top1.gif" onmouseover="this.src='http://openwetware.org/images/f/f0/Top2.gif'" onmouseout="this.src='http://openwetware.org/images/2/2a/Top1.gif'" height="150px"/></a></div> </head>

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<body> <a name="1"></a> <font face="Futura,Arial,Frutiger" font size="24px">EXPERIMENTS</font> <br> <br> <h1 class="title"><a href="high"> Highlights</a></h1>

<h1 class="sub"> TEM analysis of DNA constructs</h1>

<p class="paragraph"> Both of the DNA constructs we designed were confirmed their folding by TEM image.</p>

<div class="centerbox"> <p class="temimage"><img src="http://openwetware.org/images/c/c9/Wall_temforwiki.JPG"></p> <p class="caption">Fig.1. TEM image of the Wall.</p> </div>

<div class="centerbox"> <p class="temimage"><img src="http://openwetware.org/images/c/ce/Monomer_temforwiki.JPG";></p> <p class="caption">Fig.2. TEM image of the Motor-Monomer.</p> </div>

<h1 class="sub"> Confocal microscopic analysis of the DNA constructs in liposome</h1>

<p class="paragraph"> The DNA constructs were put into giant unilamellar vesicles (GUV). GUV were dyed with Nile Red, which appears as yellow green dots in figures, and the DNA constructs were combined with Qdots, which appear as red dots in figures.</p>

<div class="yuibox"> <center> <table cellpadding="10px"> <td><img src="http://openwetware.org/images/d/d1/Wall_sample2_003.png" height="150px"></td><td><img src="http://openwetware.org/images/e/e4/Monomer_sample1_002.png" height="150px"></td><td><img src="http://openwetware.org/images/6/6d/GUV_001_2.png" height="150px"></td> </table> </center> <p class="caption">Fig.3. Confocal fluorescence images of GUV. Left: GUV includes the Wall in its inner solution. Middle: GUV includes the Motor-Monomers in its inner solution. Right: GUV includes no DNA constructs.</p> </div>

<p class="paragraph">DNA constructs were added to the inner solution of GUV in fig.3 and fig.5 and not in fig.4 and fig.6. Red dots of the DNA constructs were appeared in GUV only in fig.3 and fig.5, we therefore confirmed the inclusion of the DNA constructs into GUV.</p>

<br>

<h1 class="sub"> TEM analysis of the simple Motor-Polymer</h1>

<div class="imagebox"> <p class="temimage"><img src="http://openwetware.org/images/7/74/Trimer_temkashiwa.png"></p> <p class="caption">Fig.7. TEM image of the simple Motor-Polymer.</p> </div>

<p class="paragraph">Though we could not achieve forming the completed Motor-Polymer, the simple Motor-Polymer using streptavidin-biotin binding was confirmed by TEM.</p> <p class="paragraph">The key factors of polymerizing are the concentration of Motor-Monomer and the addition of crowding reagents such as PEG into the solutions. Regarding concentration, high concentration of the Monomer induce the aggregation of the Monomer even in the absence of StreptAvidin, the connector fo each Monomer. We, therefore, have to keep the Monomer at moderate concentration. Howerver, efficeint polymerization of the Monomer were hindered at the low concentraiton of the Monomer. By laborious effort, we found that the addition of crowding reagents such as PEG greatly improve the formation of polymer, which might be increasing the effective concentraiton of the Monomer.</p>

<br>

<h1 class="sub"> Controlling the binding activity of the Motor-Monomer</h1> <div class="imagebox"> <p class="image"><img src="http://openwetware.org/images/8/87/Inactivated-SA-name.png"></p> <p class="caption">Fig.8. Mechanism of this experiment</p> </div>

<p class="paragraph">The binding activity of the Motor-Monomer was controlled using the difference of the streptavidin-binding strength between biotin and desthiobiotin. (Detailed explanation is in <a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#1">Design: The Motor-Monomer</a>.)</p> <p class="paragraph"> We succeeded in deactivation and reactivation of the binding activity; the deactivation using dsDNA-supported desthiobiotin and the reactivation by cutting the dsDNA with HindⅢ.</p>

<div class="centerbox"> <p class="image"><img src="http://openwetware.org/images/4/48/Inactivated-SA-cy3.png"></p> <p class="caption">Fig.9. Cy3 fluorescence image of SA-dsDNA complex by Native-PAGE.</p> </div> <div class="centerbox"> <p class="image"><img src="http://openwetware.org/images/e/e1/Inactivated-SA-cy5.png"></p> <p class="caption">Fig.10. Cy5 fluorescence image of SA-dsDNA complex by Native-PAGE.</p> </div>

<p class="paragraph"> Blocker was labeled with Cy3 and Chaser was labeled with Cy5. The band of the same position in lane 4 of fig.9 appears only in lane 7 of fig.10, which means the replacement of Blocker with Chaser happened only when HindⅢ was added. </p>

<h1 class="title"><a href="high"> More Experiments</a></h1> <p class="paragraph"> Please click buttons to read detailed experiments! </p>

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Biomod/2014/Kashiwa/Highlights

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