Biomod/2014/Kashiwa/Protocol: Difference between revisions
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<font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font> | <font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font> | ||
<br> | <br> | ||
<br> | <br> | ||
Line 209: | Line 247: | ||
<div class="CollapsibleBoxBody" id="CBoxBody1"> | <div class="CollapsibleBoxBody" id="CBoxBody1"> | ||
<ul style="list-style:none;"> | <ul style="list-style:none;"> | ||
<li> <a href=#1>1. | <li> <a href=#1> 1. The Sensing System: The Receptor </a> | ||
<ul style="list-style:none;"> | <ul style="list-style:none;"> | ||
<li> <a href="#1-1">1-1. | <li> <a href="#1-1">1-1. Preparing the components </a></li> | ||
<ul style="list-style:none;"> | <ul style="list-style:none;"> | ||
<li> <a href="#1- | <li> <a href="#1-1-1">1-1(a). Folding the Wall </a></li> | ||
<li> <a href="#1- | <li> <a href="#1-1-2">1-1(b). Producing MISTIC </a></li> | ||
<li> <a href="#1- | <li> <a href="#1-1-3">1-1(c). Designing the Activator </a></li> | ||
</ul> | </ul> | ||
</li> | </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> | </ul> | ||
</li> | </li> | ||
<li> <a href="#2">2. | |||
<li> <a href="#2">2. The Moving System: The Motor </a> | |||
<ul style="list-style:none"> | <ul style="list-style:none"> | ||
<li> <a href="#2-1">2-1. | <li> <a href="#2-1">2-1. Producing the Motor-Monomer </a> | ||
<ul style="list-style:none"> | <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> | </ul> | ||
</li> | </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> | </ul> | ||
</li> | |||
</ul> | </ul> | ||
</div> | </div> | ||
Line 249: | Line 281: | ||
<br> | <br> | ||
<a name="1-1"> </a> | |||
<br> | |||
<a name="1-1-1"> </a> | |||
<h1 class="title"><a name="contents"> 1. Developing the sensing system</a></h1> | <h1 class="title"><a name="contents"> 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">• Make the hetero-units</p> | |||
<p class="subtitle">1-1-1. Design of the Receptor hetero - units</p> | |||
<p> | |||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 40.0 µL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th> | <th>M13mp18ss</th> | ||
< | <td>18.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>Staple mix</th> | ||
<td>18 µL</td> | <td>18.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>10 × Receptor buffer¹</th> | ||
<td> | <td>4.0 µL</td> | ||
</tr> | </tr> | ||
</tbody> | |||
</table> | </table> | ||
<p> | <p class="mini">¹10 × Receptor buffer — 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"> </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> | |||
<p class=" | |||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 20.0 µL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th> | <th>10 µM Aptamers</th> | ||
< | <td>3.5 µL </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>1 µM Alpha - thrombin</th> | ||
<td> | <td>3.5 µL </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>10 × physiological buffer¹</th> | ||
<td> | <td>2.0 µ L</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>MQ</th> | ||
<td> | <td>11.0 µ L</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
< | <p class="mini">¹10 × physiological buffer — 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"> </a> | |||
< | |||
</ | |||
<p>Procedure</p> | <p class="headline">Procedure</p> | ||
<p>1) Aptamer | <p class="line">1) Aptamer solution was mixed.</p> | ||
<p>2) The | <p class="line">2) The solution was annealed from 90 °C to 24 °C, decrease by 0.4 °C / min.</p> | ||
<p>3) Thrombin-solution was added to the | <p class="line">3) Thrombin-solution was added to the solution and incubated at room temperature for 1 hour.</p> | ||
<p>4) The mixture was analyzed with Native-PAGE ( | <p class="line">4) The mixture was analyzed with Native-PAGE (200 V 25 min, 250 V 40 min).</p> | ||
<p>5) The gel was stained by EtBr for 30 minutes | <p class="line">5) The gel was stained by EtBr for 30 minutes. | ||
<p>6) The gel was observed with UV and fluorescence by LAS - 4000</p> | <p class="line">6) The gel was observed with UV and fluorescence by LAS - 4000.</p> | ||
<br> | |||
<br> | <br> | ||
<p class="subtitle">1-1-3. Emission of the Initiator</p> | |||
<p class=" | |||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 20.0 µL 25 nM)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th | <th>100 nM Biotin modified single strand J’</th> | ||
<td>5.0 µL</td> | |||
<td>5 µL</td> | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>100 nM The Mismatch modified Strand J</th> | ||
<td>5 µL</td> | <td>5.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>100 nM The Initiator</th> | ||
<td>5 µL</td> | <td>5.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>Cy5-modified Streptavidin (SA)</th> | ||
<td>5 µL</td> | <td>5.0 µL</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
< | <p class="headline">Procedure</p> | ||
<p class=" | <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> | ||
<a name="1-2-1"> </a> <br><br>< | <p class="line">2) Biotin modified single strand and SA were mixed and incubated at room temperature for 15 minutes.</p> | ||
<p class=" | <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"> </a> | |||
<br> | |||
<a name="1-2-1"> </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=" | <p class="subtitle">Ⅰ. Preparation of GUVs</p> | ||
<p> | <p class="line">GUV s were made as shown in <a href=“#2-2-1”>”Putting the Monomer into a liposome".</a></p> | ||
<br> | <br> | ||
<p class=" | <p class="subtitle">Ⅱ. Preparation of LUVs</p> | ||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 1280 µL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th> | <th>10 mg/mL POPC</th> | ||
< | <td>200.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>25 mg/mL POPG</th> | ||
<td> | <td>80.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>150 mM KCl solution</th> | ||
<td> | <td>1.0 mL</td> | ||
</tr> | </tr> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
<p> | <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> | <br> | ||
<p class=" | <p class="subtitle">Ⅲ. Penetration of the Receptor into liposomes</p> | ||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 100.0 µL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th> | <th>Liposomes (LUVs , GUVs)</th> | ||
<td>50.0 µL</td> | |||
<td> | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>Cholesterol hybridized Receptor¹</th> | ||
<td> | <td>50.0 µL</td> | ||
</tr> | </tr> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
<p class="mini">¹Cholesterol hybridized Receptor — x µM Purified Receptor, 160 x µM Cholesterol oligomer </p> | |||
<p>Procedure</p> | |||
<p> The reagents were mixed and incubated at room temperature for | <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> | <br> | ||
<p class=" | <p class="subtitle">Ⅳ. Flotation assay</p> | ||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 2.4 mL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th> | <th>The Receptor</th> | ||
< | <td>100.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>Cholesterol hybridized Receptor</th> | ||
<td>100.0 µL</td> | |||
<td>100 | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th> | <th>Liposomes</th> | ||
< | <td>100.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>2.25 M Sucrose buffer¹</th> | ||
<td> | <td>500.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>1.6 M Sucrose buffer¹</th> | ||
<td> | <td>900.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>150 mM KCl solution</th> | ||
<td> | <td>100.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>1 × Flotation buffer²</td> | ||
<td> | <td>600.0 µL</td> | ||
</tr> | </tr> | ||
</tr> | </tr> | ||
Line 568: | Line 491: | ||
</table> | </table> | ||
< | <p class="mini">¹1.6, 2.25 M Sucrose buffer — 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">²1 × Flotation buffer — 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl<sub>2</sub> | |||
</ | |||
< | |||
<p>Procedure</p> | <p class="headline">Procedure</p> | ||
<p>1) Each sample was mixed as shown below (table1).</p> | <p class="line">1) Each sample was mixed as shown below <a href="#table1">(table1)</a>.</p> | ||
<p>2) | <p class="line">2) 1.6 M Sucrose buffer was overlaid with sample mixture in centrifuge tubes (Beckman, cat#343778, 11 × 34 mm).</p> | ||
<p>3) | <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>4) 150 µL | <p class="line">4) Supernatant (each 150 µL) was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 1 × Flotation buffer (Fraction 4).</p> | ||
<p>5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).</p> | <p class="line">5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).</p> | ||
<p>6) The Intensity of fluorescence of NileRed ( | <a name="table1"></a><p class="line">6)10 µM Nile Red was added to each fraction of sample 1 and 3. (30 µL) (6 µL) 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"> | <table class="sample_04"> | ||
<caption>Table 1. Breakdown of Samples</caption> | <caption>Table 1. Breakdown of Samples</caption> | ||
Line 637: | Line 516: | ||
<tr> | <tr> | ||
<th>Cholesterol hybridized Receptor</th> | <th>Cholesterol hybridized Receptor</th> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
<td>—</td> | <td>—</td> | ||
<td>—</td> | <td>—</td> | ||
Line 646: | Line 525: | ||
<td>—</td> | <td>—</td> | ||
<td>—</td> | <td>—</td> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th>Liposome</th> | <th>Liposome</th> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
<td>—</td> | <td>—</td> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
<td>—</td> | <td>—</td> | ||
</tr> | </tr> | ||
Line 659: | Line 538: | ||
<th>150 mM aqueous KCl solution</th> | <th>150 mM aqueous KCl solution</th> | ||
<td>—</td> | <td>—</td> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
<td>—</td> | <td>—</td> | ||
<td>50 µL</td> | <td>50.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<th>2.25 M Sucrose buffer</th> | <th>2.25 M Sucrose buffer</th> | ||
<td>125 µL</td> | <td>125.0 µL</td> | ||
<td>125 µL</td> | <td>125.0 µL</td> | ||
<td>125 µL</td> | <td>125.0 µL</td> | ||
<td>125 µL</td> | <td>125.0 µL</td> | ||
</tr> | </tr> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
<a name="1-2-2"> </a> | |||
<a name="1-2-3"> </a> | |||
<br> | <br> | ||
<a name="1-2-2"> </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=" | <a name="2"> </a> | ||
< | <br> | ||
<a name="1- | <a name="2-1"> </a> | ||
<br> | |||
<a name="2-1-1"> </a> | |||
<br> | |||
<h1 class="title"><a name="contents"> 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">• Make the Moter-Monomer | |||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 60.0 µL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th | <th>Staple mix</th> | ||
<td>17.5 µL</td> | <td>17.5 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>M 13 mp 18 ss</th> | ||
<td>18.3 µL</td> | <td>18.3 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>TE¹</th> | ||
<td>18.2 µL</td> | <td>18.2 µL</td> | ||
<tr> | <tr> | ||
< | <th>10 × tile buffer²</th> | ||
<td> 6.0 µL</td> | <td> 6.0 µL</td> | ||
</tr> | </tr> | ||
Line 716: | Line 600: | ||
</table> | </table> | ||
< | <p class="mini">¹TE — 10 mM Tris - HCl (pH 8.0), 1 mM EDTA</p> | ||
<p class="mini">²10 × tile buffer — 100 mM Mg (OAc) <sub>2</sub>, 200 mM Tris-HCl (pH 7.5), .10 mM EDTA</p> | |||
< | |||
</ | |||
</ | |||
< | <a name="2-1-2"> </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> | |||
<p>Procedure</p> | |||
<p>1) The reagents were mixed.</p> | |||
<p>2) The mixture was annealed at 55 °C for 3 hours.</p> | |||
<p>3) The mixture was analyzed by 1 % agarose gel electrophoresis.</p> | |||
<p>4) The gel was stained by EtBr for 30 minutes.</p> | |||
<p>5) | |||
<br> | <br> | ||
<p class="subtitle">2-1-2. Formation of the simple Polymer</p> | |||
<p class=" | |||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f. 20.0 µL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th> | <th>Purified Monomer X</th> | ||
< | <td>10.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>Purified Monomer Y</th> | ||
<td>10.0 µL</td> | |||
<td>10 µL</td> | |||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
<p>Procedure</p> | |||
<p>1) Monomers were made as shown in “Making DNA origami monomers”.</p> | <a name="2-1-3"> </a> | ||
<p>2) Each Monomer solution was purified by spin column.</p> | |||
<p>3) The reagents | <p class="headline">Procedure</p> | ||
<p>4) The mixture was incubated at room temperature for 24 hours.</p> | <p class="line">1) Monomers were made as shown in “Making DNA origami monomers”.</p> | ||
<p>5) The mixture was analyzed by 1 % agarose gel electrophoresis (100 V, 80 min).</p> | <p class="line">2) Each Monomer solution was purified by spin column.</p> | ||
<p>6) The gel was stained by EtBr for 30 minutes.</p> | <p class="line">3) The reagents were mixed.</p> | ||
<p>7) The gel was taken a photo by LAS - 4000.</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> | <br> | ||
<a name="2-2"> </a> | |||
<p class="subtitle">2-1-3. Controlling the ring - opening polymerization </p> | |||
<a name="2-2-1"> </a> | |||
<br><br> | |||
<p class="title">2-2. The Motor in the liposome </p> | |||
<p class=" | |||
<p class="subtitle">2-2-1. Putting the Motor - Monomers into the liposome</p> | |||
<p class=" | |||
<table class="sample_01"> | <table class="sample_01"> | ||
<caption>Reagents (f.1420 µL)</caption> | |||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
<th | <th>Lipid mix ¹</th> | ||
<td>400.0 µL</td> | |||
<td>400 µL</td> | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>Glucose</th> | ||
<td>500 µL</td> | <td>500.0 µL</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
< | <th>Emulsion mix²</th> | ||
<td>520 µL</td> | <td>520.0 µL</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
< | <p class="mini">¹3 mM Lipid mix — POPC, Paraffin</p> | ||
<p class="mini">²Emulsion mix — 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"> </a> | ||
<p class="headline">Procedure</p> | |||
<p class="line">1) Glucose (500 µL) 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> | |||
<p>1) Glucose (500 µL) was taken into a tube as outer solution.</p> | |||
<p>2) Lipid mix (0.5 mM) was added on the glucose solution carefully.</p> | |||
<p>3) Emulsion mix was added on the top and rapidly centrifuged at 4 °C, 1800 rpm for 10 minutes.</p> | |||
<p>4) Centrifuged again at 4 °C, 5000 rpm for 10 minutes.</p> | |||
<p>5) The upper layer was removed and GUVs were collected by micropipette.</p> | |||
<p>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=" | <p class="subtitle">2-2-3. Controlling the ring opening polymerization in the liposome </p> | ||
<p class=" | |||
Latest revision as of 07:56, 25 October 2014
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<body> <font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font>
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<div class="CollapsibleBoxTitle"> <p class="cBoxButtons"> <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"> 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>
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<br> <a name="1-1"> </a> <br> <a name="1-1-1"> </a>
<h1 class="title"><a name="contents"> 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">• Make the hetero-units</p> <table class="sample_01"> <caption>Reagents (f. 40.0 µL)</caption> <tbody> <tr> <th>M13mp18ss</th> <td>18.0 µL</td> </tr> <tr> <th>Staple mix</th> <td>18.0 µL</td> </tr> <tr> <th>10 × Receptor buffer¹</th> <td>4.0 µL</td> </tr> </tbody> </table>
<p class="mini">¹10 × Receptor buffer — 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"> </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 µL)</caption> <tbody> <tr> <th>10 µM Aptamers</th> <td>3.5 µL </td> </tr> <tr> <th>1 µM Alpha - thrombin</th> <td>3.5 µL </td> </tr> <tr> <th>10 × physiological buffer¹</th> <td>2.0 µ L</td> </tr> <tr> <th>MQ</th> <td>11.0 µ L</td> </tr> </tbody> </table>
<p class="mini">¹10 × physiological buffer — 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"> </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 µL 25 nM)</caption> <tbody> <tr> <th>100 nM Biotin modified single strand J’</th> <td>5.0 µL</td> </tr> <tr> <th>100 nM The Mismatch modified Strand J</th> <td>5.0 µL</td> </tr> <tr> <th>100 nM The Initiator</th> <td>5.0 µL</td> </tr> <tr> <th>Cy5-modified Streptavidin (SA)</th> <td>5.0 µL</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"> </a> <br> <a name="1-2-1"> </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 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 µL)</caption> <tbody> <tr> <th>10 mg/mL POPC</th> <td>200.0 µL</td> </tr> <tr> <th>25 mg/mL POPG</th> <td>80.0 µL</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 µL)</caption>
<tbody>
<tr>
<th>Liposomes (LUVs , GUVs)</th>
<td>50.0 µL</td>
</tr>
<tr>
<th>Cholesterol hybridized Receptor¹</th>
<td>50.0 µL</td>
</tr>
</tr>
</tbody>
</table>
<p class="mini">¹Cholesterol hybridized Receptor — x µM Purified Receptor, 160 x µM 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>
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<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 µL</td> </tr> <tr> <th>Cholesterol hybridized Receptor</th> <td>100.0 µL</td> </tr> <tr> <th>Liposomes</th> <td>100.0 µL</td> </tr> <tr> <th>2.25 M Sucrose buffer¹</th> <td>500.0 µL</td> </tr> <tr> <th>1.6 M Sucrose buffer¹</th> <td>900.0 µL</td> </tr> <tr> <th>150 mM KCl solution</th> <td>100.0 µL</td> </tr> <tr> <th>1 × Flotation buffer²</td> <td>600.0 µL</td> </tr> </tr> </tbody> </table>
<p class="mini">¹1.6, 2.25 M Sucrose buffer — 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">²1 × Flotation buffer — 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 × 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 µL) was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 1 × 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 µM Nile Red was added to each fraction of sample 1 and 3. (30 µL) (6 µL) 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 µL</td> <td>50.0 µL</td> <td>—</td> <td>—</td> </tr> <tr> <th>Receptor</th> <td>—</td> <td>—</td> <td>50.0 µL</td> <td>50.0 µL</td> </tr> <tr> <th>Liposome</th> <td>50.0 µL</td> <td>—</td> <td>50.0 µL</td> <td>—</td> </tr> <tr> <th>150 mM aqueous KCl solution</th> <td>—</td> <td>50.0 µL</td> <td>—</td> <td>50.0 µL</td> </tr> <tr> <th>2.25 M Sucrose buffer</th> <td>125.0 µL</td> <td>125.0 µL</td> <td>125.0 µL</td> <td>125.0 µL</td> </tr> </tr> </tbody> </table> <a name="1-2-2"> </a> <a name="1-2-3"> </a> <br>
<a name="1-2-2"> </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"> </a> <br> <a name="2-1"> </a> <br> <a name="2-1-1"> </a> <br>
<h1 class="title"><a name="contents"> 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">• Make the Moter-Monomer
<table class="sample_01"> <caption>Reagents (f. 60.0 µL)</caption> <tbody> <tr> <th>Staple mix</th> <td>17.5 µL</td> </tr> <tr> <th>M 13 mp 18 ss</th> <td>18.3 µL</td> </tr> <tr> <th>TE¹</th> <td>18.2 µL</td> <tr> <th>10 × tile buffer²</th> <td> 6.0 µL</td> </tr> </tr> </tbody> </table>
<p class="mini">¹TE — 10 mM Tris - HCl (pH 8.0), 1 mM EDTA</p> <p class="mini">²10 × tile buffer — 100 mM Mg (OAc) <sub>2</sub>, 200 mM Tris-HCl (pH 7.5), .10 mM EDTA</p>
<a name="2-1-2"> </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 µL)</caption> <tbody> <tr> <th>Purified Monomer X</th> <td>10.0 µL</td> </tr> <tr> <th>Purified Monomer Y</th> <td>10.0 µL</td> </tr> </tbody> </table>
<a name="2-1-3"> </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"> </a>
<p class="subtitle">2-1-3. Controlling the ring - opening polymerization </p>
<a name="2-2-1"> </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 µL)</caption> <tbody> <tr> <th>Lipid mix ¹</th> <td>400.0 µL</td> </tr> <tr> <th>Glucose</th> <td>500.0 µL</td> </tr> <tr> <th>Emulsion mix²</th> <td>520.0 µL</td> </tr> </tbody> </table>
<p class="mini">¹3 mM Lipid mix — POPC, Paraffin</p> <p class="mini">²Emulsion mix — 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"> </a> <p class="headline">Procedure</p> <p class="line">1) Glucose (500 µL) 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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