Biomod/2012/TeamSendaiA/Results & Discussion
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<!-- ヘッダー --> <div id="Header"> <h1>Team Sendai</h1> <p>Tohoku University</p> </div>
<!—トップメニュー --> <ul id="Topmenu"> <li><a href=" http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai">Top</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Project">Project</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Simulation">Simulation</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion">Experiment</a></li> <li><a href=" http://openwetware.org/wiki/BIOMOD/2012/Team_Sendai/Movie">Diary</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Member ">Team</a></li> </ul> </ul>
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<h2>Contents</h2>
<ol>
<li><a href="#Plan">Experiment Plan</a></li>
<li><a href="#Selector">Selector</a></li>
<ol>
<li><a href="#SelectorDesign">Single stranded DNA sequenses</a></li>
<li><a href="#SelectorElectrophoresis">Electrophoresis</a></li>
</ol>
<li><a href="#Gate">Gate</a></li>
<ol>
<li><a href="#GateDesign">caDNAno</a></li>
<li><a href="#GateElectrophoresis">Electrophoresis</a></li>
<li><a href="#GateAFMimage">AFM image</a></li>
</ol>
<li><a href="#Membrane">Membrane</a></li>
</ol>
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<p> タイトルをもっとわかりやすいように装飾したい </p>
<!--
<a name="Selector"></a><h2>Selector</h2>
<p>
セレクターは奥に進むほどターゲット分子と配列の一致が多くなるように設計する。このことにより、筒形の構造体の内部にセレクターを生やすことで、ターゲット分子を徐々に奥に送り出すということが可能となる。</br>
セレクターにどのように番号を振り、どのように働いてくれたら嬉しいのかの図（できれば動画かな）をください。
</p>
<a name="SelectorDesign"></a> <h2> Single stranded DNA sequences</h2> <p> <img src="http://openwetware.org/images/9/97/Format_Selector_sequence.jpg" alt="selecotor sequence" width="800" height="324"/> </p>
<table style="clear:right;float:left;width:650px;border-style:solid;border-width:2px;"> <tr> <th style="width:100px;">Name</th><th style="width:450px;">Sequence</th><th style="width:100px;">Tm(°C)</th> </tr> <tr> <td style="border-style:solid;border-width:1px;">target</td><td style="border-style:solid;border-width:1px;"><font color="red">TGCAGCAGTCGTACCA</font></td><td style="border-style:solid;border-width:1px;"></td> </tr> <tr> <td style="border-style:solid;border-width:1px;">Selector1</td><td style="border-style:solid;border-width:1px;">AAAAAAAAAAAAAAAAA<font color="red">TGGTAC</font>AAAAAAAA<font color="red">GACTG</font>AAAAAAAA<font color="red">CTGCA</font></td><td style="border-style:solid;border-width:1px;">29.5</td> </tr> <tr> <td style="border-style:solid;border-width:1px;">Selector2</td><td style="border-style:solid;border-width:1px;">AAAAAAAAAAA<font color="red">TGGTAC</font>AAAA<font color="red">GCTGCA</font></td><td style="border-style:solid;border-width:1px;">35.1</td> </tr> <tr> <td style="border-style:solid;border-width:1px;">Selector3</td><td style="border-style:solid;border-width:1px;"><font color="red">TGGTACGACTGCTGCA</font></td><td style="border-style:solid;border-width:1px;">62.3</td> </tr> </table>
<p style="clear:both;"> このような配列でセレクターを作りました。</br> この構造にしたのはこういう理由です。</br> それぞれがターゲットとどのような構造を作って結合するのか絵が欲しい。配列の表、もっと小さくていい。 </p>
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<a name="SelectorElectrophoresis"></a><h2>Selector</h2> <table border="1" summary="SelectorElectrophoresis"> </p> <p> <tr> <th colspan="2">Condition</th> </tr> <tr> <td colspan="2">constant voltage: 50V</br>temperature: 4℃(in refrigerater)</br>time: 2 hours</br>SYBR Gold as a stain for 10 minutes</br>1X TAE added to Mg2+ as a buffer</td> </tr> <tr> <th>1</th> <td >Target</td> </tr> <tr> <th>2</th> <td >Selector1</td> </tr> <tr> <th>3</th> <td >Selector2</td> </tr> <tr> <th>4</th> <td >Selector3</td> </tr> <tr> <th>5</th> <td >Target + Selector1</td> </tr> <tr> <th>6</th> <td > (Target + Selector1)+Selector2</td> </tr> <tr> <th>7</th> <td > (Target + Selector1) </br>+ </br>Selector2 + Selector3</td> </tr> <tr> <th>8</th> <td > Target + Selector2</td> </tr> <tr> <th>9</th> <td > Target + Selector3</td> </tr> <tr> <th>10</th> <td >Marker</td> </tr> </table> </p>
<p> <img src="http://openwetware.org/images/a/a9/Format_9-4_erectrophoresis_new.jpg" alt="selecotor electorophoresis" width="483" height="427"/> </p>
<p> [Target is passed between Selectors]<br>
In a fridge (at 4 degree)<br>
1. hybridize the target and Selector1<br> Put a target and Selector1 in a tube and leave it for 時間<br> 2. pass the target from Selector1 to Selector2<br> After hybridizing the target and Selector1, add Selector2 and leave it for 時間<br> 3. pass the target from Selector2 to Selector3<br> After hybridizing the target and Selector2, add Selector3 and leave it for 時間<br> <br> If Selector binds to a target, its band is seen upper, and the left target is observed weakly.<br> Selector1 binds to the target (lane画像を付けて、レーンの名前). Selector1 releases the target and Selector2 catches it (laneレーン名, single stranded Selector1, and Selector2 with the target). Then Selector2 releases the target and Selector3 catches it (laneレーン名, single stranded Selector1and 2, and Selector3 with the target). Therefore, we successfully showed that target is passed between Selectors.<br> <br> [Selector in the gate catches the target]<br> 1. prepare three kinds of gates. Gate with no selector, gate with selector2, and gate with selector1 and 2.<br> 2. put each gate and a target together.<br> <br> With no selector in the gate, a target doesn’t goes in the gate(写真、laneレーンの名前). With selector2, or selector1 and 2 in the gate, a target goes into the gate (laneレーンの名前). The efficiency is the best when two selectors are planted in the gate. Thus, we concluded that selector in the gate greatly contributes to catch the target.<br> </p>
<a name="Gate"></a><h2>Gate</h2> <p>
</p> <a name="GateDesign"></a><h2>caDNAno</h2> <p> We designed the gate structure by using caDNAno. This shows our design. </p> <a name="GateElectrophoresis"></a><h2>Electrophoresis</h2> <p> We purified our gate structure to get clear image by AFM. This shows our experimental results. <p> <img src="http://openwetware.org/images/c/c2/Comparesimuvsshawn1012.png" alt="selecotor sequence" width="400" height="324"/> </p> </p> <a name="GateAFMimage"></a><h2>AFM image</h2> <p> This shows our gate image observed by AFM <p> <img src="http://openwetware.org/images/9/9e/Moyashi.png" alt="ＡＦＭ" width="483" height="427"/> </p> </br> </br> </br> </br> </br> </br> </br> </br> </br> </br> </br> </p>
<a name="Membrane"></a><h2>Membrane</h2> <p> We use fluorescein to confirm that the tube insert into the liposome. For</br> example, We put Lucifer Yellow fluorescein into big liposome and made a hole</br> using the α- Hemorijin into liposomes. Then, we observed that fluorescein was</br> flowing out from it. Alpha-hemolysin is toxin which makes a hole in the cell and is</br> often used in experiments liposome system. The way of the experience of the protein</br> a-hemolysin was dissolved in 150mM KCL, 10 mM HPES and kept at 4℃ for up to</br> 6 months. And 0.6-μL of 1 mg/mL a-hemolysin solution was added directly to a 6-</br> μL sample on the microscope slide. The figure shows that fluorescein flowing out</br> from a liposome. So, we are sure that observing fluorescein would be a confirmatory</br> experiment which our tube stuck to the liposome or not.</br> （リポソームに筒が刺さっているかどうかを確かめるために、蛍光分子を使用する。実験と</br> して、ルシファーイエロー（染色液）を大きなリポソームに入れて、リポソームにαヘモ</br> リジンを使用して穴を開け、リポソームから蛍光分子が流れ出てくるところを観察した。</br> αヘモリジンは、細胞に穴を開ける毒素の一つで、リポソーム系の実験でよく使われてい</br> る。実験結果としては、写真の通り観察できたので、筒がリポソームに刺さったかどうか</br> を確かめる方法として使用できることが分かった。ネガコンの写真 ポジコンの写真）</br>
We also can confirm that by adding hoechst to the sample, the tube is on the</br> membrane of liposome or not, and confirm that fluorescein does not leak from the</br> hole in nature.</br> （また，サンプルにhoechstを添加することにより，ＤＮＡ構造体がliposome膜上にあるか</br> 否かが判断でき，自然に開いた穴で漏れ出していないことを確認することができる．）</br>
We examined the appropriate composition of the liposome. And we decided the</br> composition; DOPC: DSPE-PEG2000: Fructose =100:1:1000.</br> （リポソームの適切な組成を調べ、DOPC：DSPE－PEG2000：Fructose = 100:1:1000 とい</br> う組成に決めた。）</br>
Lipids 0.4mM</br> Inside of Liposome is constitutedLucifer yellow(3-dihydro-1,3-dioxo-1H-benz(de)</br> isoquinoline-5,8- disulfonate dilithium 6-amino-2-((hydrazinocarbonyl)amino)-2)</br>
（DNAをヘキスト染色</br> 穴が開いていればLYが外に漏れだすはず ポジコンの実験プロトコルも書</br>
筒で穴開いた写真）</br>
</br> </br> </p>
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