Biomod/2014/Kashiwa/Experiments: Difference between revisions

Revision as of 12:16, 24 October 2014

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 <ul id="menu">
 <font face="Frutiger,Helvetica,Arial">
   <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>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROJECT</span></a>
     <ul class="sub">
       <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>
   <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>
   </li>
   <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>
     </ul>
   <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>
     <ul class="sub">
       <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>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Protocols" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROTOCOL</span></a>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">TEAM</span></a>
     <ul class="sub">
       <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>
     </ul>
   </li>
 </font>
 </ul>

</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 name="background"> Highlights</a></h1> <br>

  <p class="image"><img src="http://openwetware.org/images/9/96/Koala_005.jpg" width="300" height="213"></p>
  <p class="caption">It's the caption.<br>The agarose gel electrophoresis.</p>

</div> <br clear="right">

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        <a href="#" onclick="HideCBox('CBoxBody1');  return false;" title="折りたたみ／復元"><font color="white" font size="2">[show/hide]</font></a>
     </p>
     <p class="cBoxTitle">&nbsp; Contents：</p>
  </div>
  <div class="CollapsibleBoxBody" id="CBoxBody1">
     <ul style="list-style:none;">
        <li> <a href=#1> 1. The Sensing System: The Receptor </a>
        <ul style="list-style:none;">
           <li> <a href="#1-1">1-1. Preparing the components </a></li>
           <ul style="list-style:none;">
              <li> <a href="#1-1-1">1-1(a). Folding the Wall </a></li>
              <li> <a href="#1-1-2">1-1(b). Producing MISTIC </a></li>
              <li> <a href="#1-1-3">1-1(c). Designing the Activator </a></li>
           </ul>
           </li>
           <li> <a href="#1-2">1-2. Embedding the Wall into the liposome </a></li>
           <li> <a href="#1-3">1-3. Linking the Activator to the liposome </a></li>
           <li> <a href="#1-4">1-4. Combining the Wall and the Activator </a></li>
           <li> <a href="#1-5">1-5. Separating the Wall from the Activator </a></li>
        </ul>
        </li>
        
        <li> <a href="#2">2. The Moving System: The Motor </a>
        <ul style="list-style:none">
           <li> <a href="#2-1">2-1. Producing the Motor-Monomer </a></li>
           <li> <a href="#2-2">2-2. Deactivating and activating the binding capacity of streptavidin </a></li>
           <li> <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a></li>
        </ul>
        </li>
     </ul>
  </div>

</div>

<a name="2"></a> <br> <h1 class="title"><a name="background"> The Receptor</a></h1>

<h1 class="sub">1-1. Preparing the components </h1>

<h1 class="big">1-1(a). Folding the Wall</h1>

<p class="paragraph">In this experiment, the assembly condition of the Wall structure was optimized and results were analyzed by agarose gel electrophoresis.

The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.</p>

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(a)-1. Gel analysis of the walls annealed in different concentration of MgCl2.</p>

</div>

<p class="paragraph">The optimum results were following: <ul> <li>Optimum concentration of MgCl<sub>2</sub> is 15 mM.</li>

<li>Optimum temperature of annealing is 45.3 °C.</li>

<li>Optimum time of annealing is 5 hours.</li>

<li>Optimum concentration of NaCl is 2.5 mM.</li> </ul> </p>

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>

</div>

<p class="paragraph"> The folding is corroborated by the TEM image (Fig.1-1-1.). </p>

<h1 class="big">1-1(b). Producing MISTIC</h1> <p class="paragraph">We introduced the variant to the gene of MISTIC, a protein which penetrates the membrane for 4 times, by using Quick Change method. The purpose of this experiment is to produce mono-cysteine MISTIC. By this operation, we would be able to label arbitrary position of MISTIC when we want to label cysteine. We introduced variation to valine which was at 29th from the initiation codon.</p> <p class="paragraph">The wild type of the configuration of MISTIC was as follows.</p> <p class="paragraph"> MFCTFFEKHHR<u>KWDILLEKST</u>GVMEAMK<font color="red">V</font>TS<u>EEKEQLSTAIDRMNEGLDAFIQLY</u>NESEIDEPLIQ<u>LDDDTAELMKQARDMY</u>GQEK<u>LNEKLNTIIKQILSI</u>SVSEEGEKE</p> <p class="paragraph"> Underlined parts are considered to be the α-helix structures which penetrate membranes. A red letter show the part which we introduced variation in this experiment. </p> <p class="parahraph"> We confirmed the introduction of variant by sequencing. </p>

<h1 class="big">1-1(c). Designing the Activator</h1>

<h1 class="sub">1-2. Embedding the Wall into the liposome</h1>

<h1 class="sub">1-3. Linking the Activator to the liposome</h1>

<h1 class="sub">1-4. Combining the Wall and the Activator</h1>

<hi class="sub">1-5. Separating the Wall from the Activator</h1>

<a name="3"></a> <br> <br> <br> <h1 class="title"><a name="background"> The Motor</a></h1>

<h1 class="sub">2-1. Producing the Motor-Monomer </h1> <p>In this experiment, the assembly condition of the Motor-Monomer was optimized and the results were analyzed by agarose gel electrophoresis. The optimum conditions were confirmed by comparing migration distances of each sample. The sample of which migration distance is the longest was regarded as the optimum condition.</p>

<p>Fig.2-1-1. Gel analysis of the Motor-Monomer annealed in different concentration of MgCl2.

※新しく来たstaple によるモノマーが上手く行けばそちらのデータを使う。そうでない場合は今までの凝集してしまうデータを使う。

→Optimum concentration of MgCl2 is 15 mM

2) Optimum temperature of annealing (Annealing time: 4 hours, concentration of MgCl2: 15 mM)

（ゲル写真） Fig. Gel analysis of the Motor-Monomer annealed in different temperature.

→Optimum temperature of annealing is 45.3 ℃

3) Optimum time of annealing (Annealing temperature: 45.3 ℃, concentration of MgCl2: 15 mM)

（ゲル写真） Fig. Gel analysis of the Motor-Monomer annealed in different annealing time..

→Optimum time of annealing is 5 hours.

4) Optimum concentration of NaCl (Annealing temperature: 45.3 ℃, concentration of MgCl2: 15 mM, time: 4 hours)

（ゲル写真） Fig. Gel analysis of the Motor-Monomer annealed in different concentration of NaCl.

→Optimum concentration of NaCl is 2.5 mM

<h1 class="sub">2-2. Deactivating and activating the binding capacity of streptavidin </h1>

<h1 class="sub">2-3. Putting the Motor-Monomers into the liposome </h1> <br>

</body>

</html>

Biomod/2014/Kashiwa/Experiments: Difference between revisions

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@@ Line 197: / Line 197: @@
 <h1 class="big">1-1(b). Producing MISTIC</h1>
 <p class="paragraph">We introduced the variant to the gene of MISTIC, a protein which penetrates the membrane for 4 times, by using Quick Change method. The purpose of this experiment is to produce mono-cysteine MISTIC. By this operation, we would be able to label arbitrary position of MISTIC when we want to label cysteine. We introduced variation to valine which was at 29th from the initiation codon.</p>
-<p class="paragraph">The wild type of the configuration of MISTIC was as follows.<br>
+<p class="paragraph">The wild type of the configuration of MISTIC was as follows.</p>
-MFCTFFEKHHR<u>KWDILLEKST</u>GVMEAMK<font color="red">V</font>TS<u>EEKEQLSTAIDRMNEGLDAFIQLY</u>NESEIDEPLIQ<u>LDDDTAELMKQARDMY</u>GQEK<u>LNEKLNTIIKQILSI</u>SVSEEGEKE<br>
+<p class="paragraph">
-We made 4 types of variant, but only used this configuration.</p>
+MFCTFFEKHHR<u>KWDILLEKST</u>GVMEAMK<font color="red">V</font>TS<u>EEKEQLSTAIDRMNEGLDAFIQLY</u>NESEIDEPLIQ<u>LDDDTAELMKQARDMY</u>GQEK<u>LNEKLNTIIKQILSI</u>SVSEEGEKE</p>
+<p class="paragraph">
 Underlined parts are considered to be the α-helix structures which penetrate membranes.
 A red letter show the part which we introduced variation in this experiment.
+</p>
+<p class="parahraph">
 We confirmed the introduction of variant by sequencing.
+</p>