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<li><a href="/wiki/Biomod/2014/Sendai/temp/0821">Home</a></li>
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<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>


<font color="#ff000">赤文字は訂正</font>
<!--
<h1>Project Goal</h1>
polymerase, nickase, and restriction enzyme.
Our goal is to build the system which derives plural outputs from a single input with a time difference.<br>
 
To achieve the goal, we have developed two systems as follows.
In addition, to demonstrate these systems, we put taste substances into liposomes and effuse them.</p>
<ul>
This system aims to output signals to each input signals in order. To achieve this goal,
<li>１：the system which gets DNA which modifies the liposome and OUTPUT which starts the next reaction. (releasing system)</li>
-->
<li>２：the system which releases outputs with a time difference. (margin time system)
 
</li>
<h1>1. Programmable Output System </h1>
</ul>
<p> The purpose of this system is to release output DNA in order.
We accomplished the goal to use these two systems repeatedly.
We propose two different approaches.<br><br>
 
<h2>1:releasing system</h2>
<h2>1st Approach; Enzyme system</h2>
We have developed this system to release the output which starts the next reaction to release the DNA.
<p>Enzyme System has three processes.</p>
We used polymerases and nickases and made the KEY DNA.
1st Process:The process in which DNA Polymerase amplifies KEY DNA. (Amplifying process) <br>
By using KEY DNA, we got the DNA which modifies the liposome and the output(A) which starts the next reaction.
2nd Process:The process in which KEY DNA releases the liposome. (Releasing process) <br>
We give a full detail below.
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).


<ol>
</p>
<li>1-1: To start with, add the input to the solution which contains the template having a complementary sequence to the 3’end of the input.(A)<br>
<img src="http://openwetware.org/images/2/20/Enzyme-free_System_picture3.jpg" width="426px" height="600px">
To amplify the DNA(A1) having a complementary sequence to the 5’end of the template by using polymerases and nickases, tear the product-A off. (Fig.1)
<h3>7.</h3>
</li>
<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/9/91/Shasin2.png" width="800" height="482">
<img src="http://openwetware.org/images/d/d4/Enzyme-free_System_picture4.jpg" width=395px height="599px">
<li>次に工程１で増殖させた<font color="#0000ff">テンプレート</font><font color="#ff0000">(→テンプレートの相補DNA(A<sub>1</sub>))</font>の５’末端をリポソームをはやしたDNA分子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>
<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>
<img src="http://openwetware.org/images/3/31/Shasin3.png" width="800" height="345">
</ol>
<h2>２、（margin time system）</h2>
このシステムの目的は時間差で第一段階で得たアウトプットとは別のアウトプットを得ることである。<br>
このシステムを達成するために第一段階で得た鍵DNAと制限酵素を使うことによって最初に加えたインプットの構造を変化させた。この構造物を再びreleasing systemのインプットとして用いることで新たなアウトプットを時間差で得ることができる。詳細は以下のとおりである
<ol>
<li>第一段階で生成したA1をDNA分子Mに結合させ、DNA分子Bを生成する</li>
<li>B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（C）をアウトプットとして得る.(Fig3)</li>
<img src="http://openwetware.org/images/d/d3/Shasin4.png" width="800 height="493"">
<li>CをAと結合させ生成された構造物に制限酵素を加えインプットの３’末端を排除した構造物（D）をアウトプットとして得る。</li>
<li>４Dを他のテンプレートと結合させることによってreleasing systemとmagin time system が再び繰り返される。（Fig4）</li>
<img src="http://openwetware.org/images/5/5a/写真_2014-08-21_20_53_28.jpg">
</ol>


<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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<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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