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<li><a href="/wiki/Biomod/2014/Sendai/temp/0821">Home</a></li>
<li id="gn-home"><a href="/wiki/Biomod/2014/Sendai/temp/0821">Home</a></li>
<li><a href="/wiki/Biomod/2014/Sendai/temp/0821/Introduction">Introduction</a></li>
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<li><a href="/wiki/Biomod/2014/Sendai/temp/0821/Design">Design</a></li>
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<h1>Design</h1>
<h1>Design</h1>
１，このシステムを達成するためには以下のシステムを達成する必要がある
<p>To complete our goal, we need to develop next two systems.</p>
<ul>
1. Programmable Output System<br>
<li>インプットを加えてリポソームをアウトプットとして放出するシステム</li>
2. Taste Releasing System<br><br>
<li>同じインプットから別のアウトプットを時間差で放出するシステム</li>
 
</ul>
<!--
polymerase, nickase, and restriction enzyme.
 
In addition, to demonstrate these systems, we put taste substances into liposomes and effuse them.</p>
This system aims to output signals to each input signals in order. To achieve this goal,
-->
 
<h1>1. Programmable Output System </h1>
<p> The purpose of this system is to release output DNA in order.
We propose two different approaches.<br><br>
 
<h2>1st Approach; Enzyme system</h2>
<p>Enzyme System has three processes.</p>
1st Process:The process in which DNA Polymerase amplifies KEY DNA. (Amplifying process) <br>
2nd Process:The process in which KEY DNA releases the liposome. (Releasing process) <br>
3rd Process:The process in which restriction enzyme renews the 3’ end of the input-DNA sequence to get an output. (Renewing process)<br>
<img src="http://openwetware.org/images/9/9e/Fde1a0-01.png">
 
<h3>1.Amplifying process</h3>
 
<p>
In this process, first, domains A, B, and C in the template combine with domains A, B, and C in the input respectively. Then, polymerase copies 5’ end of the template sequence and extends input sequence. After that, nickase cleaves the end of the copied domain. Then, polymerase works at the gap created by the nickase and push out the domain A. Repeating this process again and again, we amplify the domain A. (Fig.1)
</p>
<p>
This DNA domain, we call it A1, becomes an input to following process 2 and 3.
</p>
 
<img src="http://openwetware.org/images/c/c9/Figure-01.png"><br><br>
 
<!--
 
&#9312; Domains A0, B0, and C0 in the template combine with domains A0-, B0-, and C0- in the input respectively. Input-Template complex is created.<br>
&#9313; Polymerase recognizes 3’ end of input. (Identified with dashed circle) <br>
&#9314; Extending input sequence. The sequence A1 is newly formed. <br>
&#9315; Polymerase runs away from Input-Template complex. <br>
&#9316; Nickase combines with its recognition cite. <br>
&#9317; Cleaving the end of the copied domain./ Making nick between A0 and A1. <br>
&#9318; Polymerase works from the gap (Identified with dashed circle) created by the nickase. <br>
&#9319; Running on DNA with making nucleotides and pushing out the A1. <br>
&#9320; Repeating this process again and again amplifies the number of domain A1. <br><br>
 
-->
 
<h3>2.Releasing process</h3>
<p> A1 combines with the 3’ end of the DNA which is combined with the DNA modified with liposome (output-A). Polymerase recognizes the 3’ end of the A1 and then extend the chain.
</p>
<p>
At the same time, output-A is denatured and released.
</p>
<img src="http://openwetware.org/images/1/1f/IMG_3657-02.jpg" width="735px" height="600px">
<p>A1 combines with the end of the DNA which fixes the liposome-modifying DNA (output-A).
Polymerases recognize the 3’end of the A1 and extend the chain.
At the same time, output-A is denatured and released.</p>
 
 
<h3>3.Renewing process</h3>
<img src="http://openwetware.org/images/7/7f/Figure3-01.png">
<p> When A1 combines with the gate-A, the DNA which has the recognition sequence corresponding to the restriction enzyme is released by polymerase. When this DNA combines with the structure made of input and template, a restriction enzyme activates, and cleaves the chain between A and B. By doing this, the domain near the 3’ end of the input becomes domain B. Then, the process goes back to the system-1.
</p>
<p> In this way, output-A, output-B, and output-C are released in order.</p>
 
<h2>2nd Approach; Enzyme-free System</h2>
<img src="http://openwetware.org/images/9/97/Figure_EnzymeFree-01.png">
<img src="http://openwetware.org/images/f/f6/Figure_EnzymeFree-03.png">
<img src="http://openwetware.org/images/8/89/Figure_EnzymeFree-04.png">
<p>This approach is inspired by seesaw gate (Lulu Qian et.al, 2011).We prepared an Input-DNA sequence, a trigger-DNA sequence, three kinds of DNA sequence (fuel, input, gate), and three kinds of double strand DNA with liposome (figure 1). We assembled a circuit with seesaw gate mechanism and those DNA sequences.</p>
<img src="http://openwetware.org/images/7/79/Figure_EnzymeFree-02.png">
<p>Reactions as follows : </p>
<h3>1.</h3>
<p>First, add input to trigger-, fuel-, gate-DNA sequence, and three kinds of double strand DNA with a liposome solution. Then, input reacts with trigger, and DNA (1) peels off.</p>
<h3>2.</h3>
<p>DNA (1) which peeled off at process 1 reacts with double strand DNA with a liposome, and effuse a single strand DNA with a liposome. This reaction takes place before process 3 because toe hold is longer than gate toe hold as seesaw gate’s threshold reaction.</p>
<h3>3.</h3>
<p>On the other hand, remaining DNA(1) reacts with the gate, and DNA(2) is released from the gate.
Then, the gate free from DNA(2) and the part of DNA(1) couples complementarily .
As a result, we get double-strand DNA(3) from them.</p>
<h3>4.</h3>
<p>Double-strand DNA and fuel react, and DNA(2) is made.</p>
<h3>5.</h3>
<p>Repeating the method3 and method4 again and again, the amount of DNA(2) increases.
(If you want to know about method1~5, refer Figure2.)
 
</p>
<img src="http://openwetware.org/images/4/42/Enzyme-free_System_picture2.jpg" width="441px" height="599px">
<h3>6.</h3>
<p>DNA(2) plays the role as a trigger, reacts with the input, induces the same reaction as the one previously described.
At this point, we get DNA(4) in the same manner as DNA(2).
 
</p>
<img src="http://openwetware.org/images/2/20/Enzyme-free_System_picture3.jpg" width="426px" height="600px">
<h3>7.</h3>
<p>DNA(4) plays the role as a trigger, reacts with the input, induces the same reaction as the one previously described.(Figure4)</p>
<img src="http://openwetware.org/images/d/d4/Enzyme-free_System_picture4.jpg" width=395px height="599px">
<p>Taking advantage of such reactions, we are convinced of outputting the targeting output in order. (taking advantage of such reactions → by these reactions)</p>


2,第一段階：リポソーム放出回路
<h2>2. Taste releasing system </h2>
このシステムの目的はリポソームを放出し次の反応につなげるためのインプットを放出するためである。
<p>
<ol>
The purpose of this system is to encapsulate taste substances in liposome and to release liposome attached to substrate.
<li>はじめにテンプレートが入った溶液にインプットを加え結合させる。結合させてできた生成物をポリメ　　　　　 　ニッカーゼを用いてテンプレートの５’末端を増幅する。（Fig1）</li>
<li>次に工程１で増殖させたテンプレートの５’末端をリポソーム修飾DNAと結合させポリメラーゼを用 　いてディナチュレーションさせリポソーム付きDNAをアウトプットとして得る。（Fig2）</li>
</ol>


３,第二段階：時間差放出システム
The DNA which has the complementary sequence (comp DNA) to the Output is sprouted on agarose gel. They are hybridized each other. When A1 reacts with comp DNA, the output is separated from comp DNA, then the liposome is released from the agarose gel, and taste substances are diffused.
このシステムの目的は第一段階で用いたINPUTから時間差で第一段階で放出したアウトプットとは別のアウトプットを放出するためのものである。
</p>
<ol>
<li>第一段階で生成したテンプレートの５’末端をDNA回路Mに結合させる</li>
<li>工程１で生成された構造物にポリメラーゼを加えインプットの３’末端に結合するDNAをアウトプットとして得る。（Fig3）</li>
<li></li>
</ol>


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Latest revision as of 08:31, 27 August 2014

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<body> <div id="globalnav" class="design"> <ul> <li id="gn-home"><a href="/wiki/Biomod/2014/Sendai/temp/0821">Home</a></li> <li id="gn-intro"><a href="/wiki/Biomod/2014/Sendai/temp/0821/Introduction">Introduction</a></li> <li id="gn-design"><a href="/wiki/Biomod/2014/Sendai/temp/0821/Design">Design</a></li> <li id="gn-simu"><a href="/wiki/Biomod/2014/Sendai/temp/0821/Simulation">Simulation</a></li> <li id="gn-xp"><a href="/wiki/Biomod/2014/Sendai/temp/0821/Experiment">Experiment</a></li> <!--<li id="gn-protocol"><a href="/wiki/Biomod/2014/Sendai/temp/0821/Protocol">Protocol</a></li>--> <li id="gn-dis"><a href="/wiki/Biomod/2014/Sendai/temp/0821/Discussion">Discussion</a></li> <li id="gn-team"><a href="/wiki/Biomod/2014/Sendai/temp/0821/Team">Team</a></li> <li id="gn-end"><a href="#"></a></li> </ul> </div> <div id="main"> <h1>Design</h1> <p>To complete our goal, we need to develop next two systems.</p> 1. Programmable Output System<br> 2. Taste Releasing System<br><br>

<!-- polymerase, nickase, and restriction enzyme.

In addition, to demonstrate these systems, we put taste substances into liposomes and effuse them.</p> This system aims to output signals to each input signals in order. To achieve this goal, -->

<h1>1. Programmable Output System </h1> <p> The purpose of this system is to release output DNA in order. We propose two different approaches.<br><br>

<h2>1st Approach; Enzyme system</h2> <p>Enzyme System has three processes.</p> 1st Process:The process in which DNA Polymerase amplifies KEY DNA. (Amplifying process) <br> 2nd Process:The process in which KEY DNA releases the liposome. (Releasing process) <br> 3rd Process:The process in which restriction enzyme renews the 3’ end of the input-DNA sequence to get an output. (Renewing process)<br> <img src="http://openwetware.org/images/9/9e/Fde1a0-01.png">

<h3>1.Amplifying process</h3>

<p> In this process, first, domains A, B, and C in the template combine with domains A, B, and C in the input respectively. Then, polymerase copies 5’ end of the template sequence and extends input sequence. After that, nickase cleaves the end of the copied domain. Then, polymerase works at the gap created by the nickase and push out the domain A. Repeating this process again and again, we amplify the domain A. (Fig.1) </p> <p> This DNA domain, we call it A1, becomes an input to following process 2 and 3. </p>

<img src="http://openwetware.org/images/c/c9/Figure-01.png"><br><br>

<!--

&#9312; Domains A0, B0, and C0 in the template combine with domains A0-, B0-, and C0- in the input respectively. Input-Template complex is created.<br> &#9313; Polymerase recognizes 3’ end of input. (Identified with dashed circle) <br> &#9314; Extending input sequence. The sequence A1 is newly formed. <br> &#9315; Polymerase runs away from Input-Template complex. <br> &#9316; Nickase combines with its recognition cite. <br> &#9317; Cleaving the end of the copied domain./ Making nick between A0 and A1. <br> &#9318; Polymerase works from the gap (Identified with dashed circle) created by the nickase. <br> &#9319; Running on DNA with making nucleotides and pushing out the A1. <br> &#9320; Repeating this process again and again amplifies the number of domain A1. <br><br>

-->

<h3>2.Releasing process</h3> <p> A1 combines with the 3’ end of the DNA which is combined with the DNA modified with liposome (output-A). Polymerase recognizes the 3’ end of the A1 and then extend the chain. </p> <p> At the same time, output-A is denatured and released. </p> <img src="http://openwetware.org/images/1/1f/IMG_3657-02.jpg" width="735px" height="600px"> <p>A1 combines with the end of the DNA which fixes the liposome-modifying DNA (output-A). Polymerases recognize the 3’end of the A1 and extend the chain. At the same time, output-A is denatured and released.</p>


<h3>3.Renewing process</h3> <img src="http://openwetware.org/images/7/7f/Figure3-01.png"> <p> When A1 combines with the gate-A, the DNA which has the recognition sequence corresponding to the restriction enzyme is released by polymerase. When this DNA combines with the structure made of input and template, a restriction enzyme activates, and cleaves the chain between A and B. By doing this, the domain near the 3’ end of the input becomes domain B. Then, the process goes back to the system-1. </p> <p> In this way, output-A, output-B, and output-C are released in order.</p>

<h2>2nd Approach; Enzyme-free System</h2> <img src="http://openwetware.org/images/9/97/Figure_EnzymeFree-01.png"> <img src="http://openwetware.org/images/f/f6/Figure_EnzymeFree-03.png"> <img src="http://openwetware.org/images/8/89/Figure_EnzymeFree-04.png"> <p>This approach is inspired by seesaw gate (Lulu Qian et.al, 2011).We prepared an Input-DNA sequence, a trigger-DNA sequence, three kinds of DNA sequence (fuel, input, gate), and three kinds of double strand DNA with liposome (figure 1). We assembled a circuit with seesaw gate mechanism and those DNA sequences.</p> <img src="http://openwetware.org/images/7/79/Figure_EnzymeFree-02.png"> <p>Reactions as follows : </p> <h3>1.</h3> <p>First, add input to trigger-, fuel-, gate-DNA sequence, and three kinds of double strand DNA with a liposome solution. Then, input reacts with trigger, and DNA (1) peels off.</p> <h3>2.</h3> <p>DNA (1) which peeled off at process 1 reacts with double strand DNA with a liposome, and effuse a single strand DNA with a liposome. This reaction takes place before process 3 because toe hold is longer than gate toe hold as seesaw gate’s threshold reaction.</p> <h3>3.</h3> <p>On the other hand, remaining DNA(1) reacts with the gate, and DNA(2) is released from the gate. Then, the gate free from DNA(2) and the part of DNA(1) couples complementarily . As a result, we get double-strand DNA(3) from them.</p> <h3>4.</h3> <p>Double-strand DNA and fuel react, and DNA(2) is made.</p> <h3>5.</h3> <p>Repeating the method3 and method4 again and again, the amount of DNA(2) increases. (If you want to know about method1~5, refer Figure2.)

</p> <img src="http://openwetware.org/images/4/42/Enzyme-free_System_picture2.jpg" width="441px" height="599px"> <h3>6.</h3> <p>DNA(2) plays the role as a trigger, reacts with the input, induces the same reaction as the one previously described. At this point, we get DNA(4) in the same manner as DNA(2).

</p> <img src="http://openwetware.org/images/2/20/Enzyme-free_System_picture3.jpg" width="426px" height="600px"> <h3>7.</h3> <p>DNA(4) plays the role as a trigger, reacts with the input, induces the same reaction as the one previously described.(Figure4)</p> <img src="http://openwetware.org/images/d/d4/Enzyme-free_System_picture4.jpg" width=395px height="599px"> <p>Taking advantage of such reactions, we are convinced of outputting the targeting output in order. (taking advantage of such reactions → by these reactions)</p>

<h2>2. Taste releasing system </h2> <p> The purpose of this system is to encapsulate taste substances in liposome and to release liposome attached to substrate.

The DNA which has the complementary sequence (comp DNA) to the Output is sprouted on agarose gel. They are hybridized each other. When A1 reacts with comp DNA, the output is separated from comp DNA, then the liposome is released from the agarose gel, and taste substances are diffused. </p>

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