Biomod/2014/Kashiwa/Protocol

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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>
     <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><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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">DESIGN</span></a>
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       <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>
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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>
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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;">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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<body> <font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font>

<br> <br> <div class="CollapsibleBox" id="CBoxCover1">

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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>
           <ul style="list-style:none">
             <li> <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a></li>
             <li> <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a></li>
             <li> <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a></li>
           </ul>
           </li>
           <li> <a href="#2-2">2-2. Deactivating and reactivating 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>

<br> <a name="1-1">&nbsp;</a> <br> <a name="1-1-1">&nbsp;</a>

<h1 class="title"><a name="contents">&nbsp;1. Developing the sensing system</a></h1> <p class="title">1-1. The Receptor by itself</p>

<p class="subtitle">1-1-1. Design of the Receptor hetero-units</p> <p class="headline">&bull; Make the hetero-units</p> <table class="sample_01"> <caption>Reagents (f. 40.0 &micro;L)</caption> <tbody> <tr> <th>M13mp18ss</th> <td>18.0 &micro;L</td> </tr> <tr> <th>Staple mix</th> <td>18.0 &micro;L</td> </tr> <tr> <th>10 &times; Receptor buffer&sup1;</th> <td>4.0 &micro;L</td> </tr> </tbody> </table>


<p class="mini">&sup1;10 &times; Receptor buffer &mdash; 50 mM Tris (HCl pH 7.5), 10 mM EDTA-Na (pH 8),<br> 100 mM MgCl<sub>2</sub>, 500 mM NaCl </p>

<a name="1-1-2">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) The solution was mixed.</p> <p class="line">2) The mixture was annealed at 47.5 °C for 4 hours.</p> <p class="line">3) The mixture was purified by spin column.</p> <p class="line">4) The mixture was analyzed by 1% agarose gel electrophoresis (100V, 40 min).</p> <br> <br>

<p class="subtitle">1-1-2. Dimerization mechanism of the Receptor</p> <table class="sample_01"> <caption>Reagents (f. 20.0 &micro;L)</caption> <tbody> <tr> <th>10 &micro;M Aptamers</th> <td>3.5 &micro;L </td> </tr> <tr> <th>1 &micro;M Alpha - thrombin</th> <td>3.5 &micro;L </td> </tr> <tr> <th>10 &times; physiological buffer&sup1;</th> <td>2.0 &micro; L</td> </tr> <tr> <th>MQ</th> <td>11.0 &micro; L</td> </tr> </tbody> </table>

<p class="mini">&sup1;10 &times; physiological buffer &mdash; 200 mM Tris - HCl (pH 7.5), 1.4 M NaCl, 50 mM KCl, <br> 10 mM CaCl <sub>2</sub>, 10 mM MgCl <sub>2</sub>, 5% (v/v) Glycerol</p>

<a name="1-1-3">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) Aptamer solution was mixed.</p> <p class="line">2) The solution was annealed from 90 °C to 24 °C, decrease by 0.4 °C / min.</p> <p class="line">3) Thrombin-solution was added to the solution and incubated at room temperature for 1 hour.</p> <p class="line">4) The mixture was analyzed with Native-PAGE (200 V 25 min, 250 V 40 min).</p> <p class="line">5) The gel was stained by EtBr for 30 minutes. <p class="line">6) The gel was observed with UV and fluorescence by LAS - 4000.</p> <br> <br>

<p class="subtitle">1-1-3. Emission of the Initiator</p> <table class="sample_01"> <caption>Reagents (f. 20.0 &micro;L 25 nM)</caption> <tbody> <tr> <th>100 nM Biotin modified single strand J’</th> <td>5.0 &micro;L</td> </tr> <tr> <th>100 nM The Mismatch modified Strand J</th> <td>5.0 &micro;L</td> </tr> <tr> <th>100 nM The Initiator</th> <td>5.0 &micro;L</td> </tr> <tr> <th>Cy5-modified Streptavidin (SA)</th> <td>5.0 &micro;L</td> </tr> </tbody> </table>


<p class="headline">Procedure</p> <p class="line">1) Strand J and Initiator were mixed and annealed at 85 °C,67 °C,50 °C,25 °C for 5 minutes each (mismatch factor 20 %).</p> <p class="line">2) Biotin modified single strand and SA were mixed and incubated at room temperature for 15 minutes.</p> <p class="line">3) The reagents were mixed and incubated at room temperature for 1 hour.</p> <p class="line">4) The mixture was analyzed with Native - PAGE (200 V 40 min, 250 V 40 min).</p> <p class="line">5) The gel was stained by EtBr for 30 minutes</p> <p class="line">6) The gel was observed with UV and fluorescence by LAS - 4000.</p> <a name="1-2">&nbsp;</a> <br> <a name="1-2-1">&nbsp;</a> <br> <br>

<p class="title">1-2. The Receptor on the liposome</p> <p class="subtitle">1-2-1. Penetration of the Receptor hetero-units to the liposome</p>

<p class="subtitle">Ⅰ. Preparation of GUVs</p> <p class="line">GUV&nbsp;s were made as shown in <a href=“#2-2-1”>”Putting the Monomer into a liposome".</a></p> <br>

<p class="subtitle">Ⅱ. Preparation of LUVs</p> <table class="sample_01"> <caption>Reagents (f. 1280 &micro;L)</caption> <tbody> <tr> <th>10 mg/mL POPC</th> <td>200.0 &micro;L</td> </tr> <tr> <th>25 mg/mL POPG</th> <td>80.0 &micro;L</td> </tr> <tr> <th>150 mM KCl solution</th> <td>1.0 mL</td> </tr> </tr> </tbody> </table> <p class="headline">Procedure </p> <p class="line">1) A lipid film was formed by evaporating POPC and POPG in a tube. </p> <p class="line">2) The tube was kept under vacuum overnight to evaporate remaining chloroform. </p> <p class="line">3) The lipid film was resuspended in 1 mL of 150 mM KCl solution. <br>Temperature was controlled to be 40 °C during suspension. </p> <p class="line">4) The solution was kept at 4 °C or -80°C and sample was sonicated before usage.</p> <br>


<p class="subtitle">Ⅲ. Penetration of the Receptor into liposomes</p> <table class="sample_01"> <caption>Reagents (f. 100.0 &micro;L)</caption> <tbody> <tr> <th>Liposomes (LUVs , GUVs)</th> <td>50.0 &micro;L</td> </tr> <tr> <th>Cholesterol hybridized Receptor&sup1;</th> <td>50.0 &micro;L</td> </tr> </tr> </tbody> </table> <p class="mini">&sup1;Cholesterol hybridized Receptor &mdash; x &micro;M Purified Receptor, 160 x &micro;M Cholesterol oligomer </p>


<p class="headline">Procedure</p> <p class="line">1)Purified Receptor and cholesterol oligomer were mixed and incubated at room temperature for 60 minutes.</p> <p class="line">2)The reagents were mixed and incubated at room temperature for 30 minutes.</p> <br>


<p class="subtitle">Ⅳ. Flotation assay</p>

<table class="sample_01"> <caption>Reagents (f. 2.4 mL)</caption> <tbody> <tr> <th>The Receptor</th> <td>100.0 &micro;L</td> </tr> <tr> <th>Cholesterol hybridized Receptor</th> <td>100.0 &micro;L</td> </tr> <tr> <th>Liposomes</th> <td>100.0 &micro;L</td> </tr> <tr> <th>2.25 M Sucrose buffer&sup1;</th> <td>500.0 &micro;L</td> </tr> <tr> <th>1.6 M Sucrose buffer&sup1;</th> <td>900.0 &micro;L</td> </tr> <tr> <th>150 mM KCl solution</th> <td>100.0 &micro;L</td> </tr> <tr> <th>1 &times; Flotation buffer&sup2;</td> <td>600.0 &micro;L</td> </tr> </tr> </tbody> </table>

<p class="mini">&sup1;1.6, 2.25 M Sucrose buffer &mdash; 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl<sub>2</sub>, 1.6, 2.25 M Sucrose </p> <p class="mini">&sup2;1 &times; Flotation buffer &mdash; 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl<sub>2</sub>


<p class="headline">Procedure</p> <p class="line">1) Each sample was mixed as shown below <a href="#table1">(table1)</a>.</p> <p class="line">2) 1.6 M Sucrose buffer was overlaid with sample mixture in centrifuge tubes (Beckman, cat#343778, 11 &times; 34 mm).</p> <p class="line">3) Each sample was centrifuged for 16 minutes at 100 krpm at 4 °C using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).</p> <p class="line">4) Supernatant (each 150 &micro;L) was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 1 &times; Flotation buffer (Fraction 4).</p> <p class="line">5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).</p> <a name="table1"></a><p class="line">6)10 &micro;M Nile Red was added to each fraction of sample 1 and 3. (30 &micro;L) (6 &micro;L) The Intensity of fluorescence of NileRed (liposomes) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposomes in each Fraction.</p>

<br> <table class="sample_04"> <caption>Table 1. Breakdown of Samples</caption> <tbody> <tr> <th>sample No.</th> <td>1</td> <td>2</td> <td>3</td> <td>4</td> </tr> <tr> <th>Cholesterol hybridized Receptor</th> <td>50.0 &micro;L</td> <td>50.0 &micro;L</td> <td>&mdash;</td> <td>&mdash;</td> </tr> <tr> <th>Receptor</th> <td>&mdash;</td> <td>&mdash;</td> <td>50.0 &micro;L</td> <td>50.0 &micro;L</td> </tr> <tr> <th>Liposome</th> <td>50.0 &micro;L</td> <td>&mdash;</td> <td>50.0 &micro;L</td> <td>&mdash;</td> </tr> <tr> <th>150 mM aqueous KCl solution</th> <td>&mdash;</td> <td>50.0 &micro;L</td> <td>&mdash;</td> <td>50.0 &micro;L</td> </tr> <tr> <th>2.25 M Sucrose buffer</th> <td>125.0 &micro;L</td> <td>125.0 &micro;L</td> <td>125.0 &micro;L</td> <td>125.0 &micro;L</td> </tr> </tr> </tbody> </table> <a name="1-2-2">&nbsp;</a> <a name="1-2-3">&nbsp;</a> <br>


<a name="1-2-2">&nbsp;</a> <p class="subtitle">1-2-2. Dimerization mechanism of the Receptor on the liposome</p>

<p class="subtitle">1-2-3. Emission of the initiator in the liposome </p>


<a name="2">&nbsp;</a> <br> <a name="2-1">&nbsp;</a> <br> <a name="2-1-1">&nbsp;</a> <br>

<h1 class="title"><a name="contents">&nbsp;2. Developing the moving system</a></h1>

<p class="title">2-1. The Motor by itself</p>

<p class="subtitle">2-1-1. Design of the Motor-Monomer </p> <p class="headline">&bull; Make the Moter-Monomer

<table class="sample_01"> <caption>Reagents (f. 60.0 &micro;L)</caption> <tbody> <tr> <th>Staple mix</th> <td>17.5 &micro;L</td> </tr> <tr> <th>M 13 mp 18 ss</th> <td>18.3 &micro;L</td> </tr> <tr> <th>TE&sup1;</th> <td>18.2 &micro;L</td> <tr> <th>10 &times; tile buffer&sup2;</th> <td> 6.0 &micro;L</td> </tr> </tr> </tbody> </table>

<p class="mini">&sup1;TE &mdash; 10 mM Tris - HCl (pH 8.0), 1 mM EDTA</p> <p class="mini">&sup2;10 &times; tile buffer &mdash; 100 mM Mg (OAc) <sub>2</sub>, 200 mM Tris-HCl (pH 7.5), .10 mM EDTA</p>

<a name="2-1-2">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) The reagents were mixed.</p> <p class="line">2) The mixture was annealed at 55 °C for 3 hours.</p> <p class="line">3) The mixture was analyzed by 1 % agarose gel electrophoresis.</p> <p class="line">4) The gel was stained by EtBr for 30 minutes.</p> <p class="line">5) A photo of the gel was taken by LAS - 4000.</p> <br> <br>

<p class="subtitle">2-1-2. Formation of the simple Polymer</p> <table class="sample_01"> <caption>Reagents (f. 20.0 &micro;L)</caption> <tbody> <tr> <th>Purified Monomer X</th> <td>10.0 &micro;L</td> </tr> <tr> <th>Purified Monomer Y</th> <td>10.0 &micro;L</td> </tr> </tbody> </table>

<a name="2-1-3">&nbsp;</a>

<p class="headline">Procedure</p> <p class="line">1) Monomers were made as shown in “Making DNA origami monomers”.</p> <p class="line">2) Each Monomer solution was purified by spin column.</p> <p class="line">3) The reagents were mixed.</p> <p class="line">4) The mixture was incubated at room temperature for 24 hours.</p> <p class="line">5) The mixture was analyzed by 1 % agarose gel electrophoresis (100 V, 80 min).</p> <p class="line">6) The gel was stained by EtBr for 30 minutes.</p> <p class="line">7) The gel was taken a photo by LAS - 4000.</p> <br> <br>


<a name="2-2">&nbsp;</a> <p class="subtitle">2-1-3. Controlling the ring - opening polymerization </p> <a name="2-2-1">&nbsp;</a> <br><br>

<p class="title">2-2. The Motor in the liposome </p>

<p class="subtitle">2-2-1. Putting the Motor - Monomers into the liposome</p> <table class="sample_01"> <caption>Reagents (f.1420 &micro;L)</caption> <tbody> <tr> <th>Lipid mix &sup1;</th> <td>400.0 &micro;L</td> </tr> <tr> <th>Glucose</th> <td>500.0 &micro;L</td> </tr> <tr> <th>Emulsion mix&sup2;</th> <td>520.0 &micro;L</td> </tr> </tbody> </table>

<p class="mini">&sup1;3 mM Lipid mix &mdash; POPC, Paraffin</p> <p class="mini">&sup2;Emulsion mix &mdash; 0.5 mM Lipid mix, 50 mM HEPES, 10 mM EDTA,<br>400 mM Sucrose,Pyranine, The Motor-Monomers</p>

<a name="2-2-2">&nbsp;</a> <p class="headline">Procedure</p> <p class="line">1) Glucose (500 &micro;L) was taken into a tube as outer solution.</p> <p class="line">2) Lipid mix (0.5 mM) was added on the glucose solution carefully.</p> <p class="line">3) Emulsion mix was added on the top and rapidly centrifuged at 4 °C, 1800 rpm for 10 minutes.</p> <p class="line">4) Centrifuged again at 4 °C, 5000 rpm for 10 minutes.</p> <p class="line">5) The upper layer was removed and GUVs were collected by micropipette.</p> <p class="line">6) The GUVs were stained by Nile red and observed with a confocal microscope.</p> <br> <br>


<p class="subtitle">2-2-2. Formation of the simple Polymer in the liposome </p>

<p class="subtitle">2-2-3. Controlling the ring opening polymerization in the liposome </p>


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