Biomod/2012/TeamSendai/Design: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
 
(110 intermediate revisions by 4 users not shown)
Line 1: Line 1:
<html>
<html>
<head>
    <title>Team Sendai Top</title>
    <script type="text/javascript" src="http://code.jquery.com/jquery-latest.min.js"></script>
    <script type="text/javascript">
        $(function() {
          if ($.browser.msie && $.browser.version.substr(0,1)<7)
          {
$('li').has('ul').mouseover(function(){
$(this).children('ul').show();
}).mouseout(function(){
$(this).children('ul').hide();
})
          }
        });       
    </script>
<style>
#Container
{
width: 960px;
margin: 40px auto;
}


/* Main menu */
<head><style>#column-one { display:none; width:0px;}
#menu
.container{background-color: #f5f5f5; margin-top:50px}
{
.OWWNBcpCurrentDateFilled {display: none;}
width: 100%;
#content {width: 0px; margin: 0 auto auto 0;  padding: 1em 1em 1em 1em; align: center;}
margin: 0;
#column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}
padding: 10px 0 0 0;
.firstHeading {display:none; width:0px;}
list-style: none;
#globalWrapper{width:1280px; margin:auto}
background: #111;
body {background: #F0F0F0 !important;}
background: -moz-linear-gradient(#444, #111);  
#column-one {display:none; width:0px;background-color: #f0f0f0;}
    background: -webkit-gradient(linear,left bottom,left top,color-stop(0, #111),color-stop(1, #444));
#content{border:none;margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: 800px;background-color: #f0f0f0; }
background: -webkit-linear-gradient(#444, #111);
.container{ width:1280px; margin: auto; background-color: #f0f0f0; text-align:justify; font-family: helvetica, arial, sans-serif; color:#f0f0f0; margin-top:25px; }
background: -o-linear-gradient(#444, #111);
#bodyContent{ width: 1267px; align: center; background-color: #f0f0f0;}
background: -ms-linear-gradient(#444, #111);
#column-content{width: 1280px;background-color: #f0f0f0;}
background: linear-gradient(#444, #111);
.firstHeading { display:none;width:0px;background-color: #f0f0f0;}
-moz-border-radius: 50px;
#header{position: center; width: 800px;background-color: #f0f0f0;}
border-radius: 50px;
#footer{position: center; width:1280px;}
-moz-box-shadow: 0 2px 1px #9c9c9c;
</style><header>
-webkit-box-shadow: 0 2px 1px #9c9c9c;
box-shadow: 0 2px 1px #9c9c9c;
}


#menu li
</html>
{
float: left;
padding: 0 0 10px 0;
position: relative;
line-height: 0;
}


#menu a
{
float: left;
height: 25px;
padding: 0 25px;
color: #999;
text-transform: uppercase;
font: bold 12px/25px Arial, Helvetica;
text-decoration: none;
text-shadow: 0 1px 0 #000;
}


#menu li:hover > a
{
color: #fafafa;
}


*html #menu li a:hover /* IE6 */
{
color: #fafafa;
}


#menu li:hover > ul
{{:Biomod/2012/Tohoku/Team_Sendai/header}}
{
display: block;
}


/* Sub-menu */
#menu ul
{
    list-style: none;
    margin: 0;
    padding: 0;   
    display: none;
    position: absolute;
    top: 35px;
    left: 0;
    z-index: 99999;   
    background: #444;
    background: -moz-linear-gradient(#444, #111);
    background: -webkit-gradient(linear,left bottom,left top,color-stop(0, #111),color-stop(1, #444));
    background: -webkit-linear-gradient(#444, #111);   
    background: -o-linear-gradient(#444, #111);
    background: -ms-linear-gradient(#444, #111);
    background: linear-gradient(#444, #111);
    -moz-box-shadow: 0 0 2px rgba(255,255,255,.5);
    -webkit-box-shadow: 0 0 2px rgba(255,255,255,.5);
    box-shadow: 0 0 2px rgba(255,255,255,.5);
    -moz-border-radius: 5px;
    border-radius: 5px;
}
#menu ul ul
{
  top: 0;
  left: 150px;
}
#menu ul li
{
    float: none;
    margin: 0;
    padding: 0;
    display: block; 
    -moz-box-shadow: 0 1px 0 #111111, 0 2px 0 #777777;
    -webkit-box-shadow: 0 1px 0 #111111, 0 2px 0 #777777;
    box-shadow: 0 1px 0 #111111, 0 2px 0 #777777;
}
#menu ul li:last-child
{ 
    -moz-box-shadow: none;
    -webkit-box-shadow: none;
    box-shadow: none;   
}
#menu ul a
{   
    padding: 10px;
height: 10px;
width: 130px;
height: auto;
    line-height: 1;
    display: block;
    white-space: nowrap;
    float: none;
text-transform: none;
}
*html #menu ul a /* IE6 */
{   
height: 10px;
}
*:first-child+html #menu ul a /* IE7 */
{   
height: 10px;
}
#menu ul a:hover
{
    background: #0186ba;
background: -moz-linear-gradient(#04acec,  #0186ba);
background: -webkit-gradient(linear, left top, left bottom, from(#04acec), to(#0186ba));
background: -webkit-linear-gradient(#04acec,  #0186ba);
background: -o-linear-gradient(#04acec,  #0186ba);
background: -ms-linear-gradient(#04acec,  #0186ba);
background: linear-gradient(#04acec,  #0186ba);
}
#menu ul li:first-child > a
{
    -moz-border-radius: 5px 5px 0 0;
    border-radius: 5px 5px 0 0;
}
#menu ul li:first-child > a:after
{
    content: '';
    position: absolute;
    left: 30px;
    top: -8px;
    width: 0;
    height: 0;
    border-left: 5px solid transparent;
    border-right: 5px solid transparent;
    border-bottom: 8px solid #444;
}
#menu ul ul li:first-child a:after
{
    left: -8px;
    top: 12px;
    width: 0;
    height: 0;
    border-left: 0;
    border-bottom: 5px solid transparent;
    border-top: 5px solid transparent;
    border-right: 8px solid #444;
}
#menu ul li:first-child a:hover:after
{
    border-bottom-color: #04acec;
}
#menu ul ul li:first-child a:hover:after
{
    border-right-color: #04acec;
    border-bottom-color: transparent;
}


<div id="Container">
   
<!-- コンテンツ -->
<div id="Content">


#menu ul li:last-child > a
<h1>Design</h1>
{
<h2>Gate</h2>
    -moz-border-radius: 0 0 5px 5px;
<h3>Size / Structure</h3>
    border-radius: 0 0 5px 5px;
What structure is most suitable for the Gate? The Gate has to connect inside and outside of the cell. So we decided to apply a hexagonal tube nanostructure made of DNA origami. We refer "A logic-gated nanorobot for targeted transport of molecular payloads" (SM Douglas, I Bachelet, GM Church - Science Signalling, 2012) for the hexagonal tube structure of DNA origami.<br>
}
Next, we made a simulation in order to examine the size of the structure. The size of the tube must be small enough not to pass freely through anything. However, it must be large enough to pass through the desired product. The gate which made of DNA origami has negative electric charge. So if the gate is too small, target can't enter the Gate. According to simulation, our Gate size determined 24*24*33nm. This size is suitable to transport the target.<br>
{{-}}


/* Clear floated elements */
<h3>DNA origami</h3>
#menu:after
We used caDNAno to design the hexagonal tube structure. This Gate tube is made from 6792bp M13mp18 and a lot of single stranded DNAs. And the Gate has double hexagonal structure because I think that is stronger than single hexagonal structure.
{
visibility: hidden;
display: block;
font-size: 0;
content: " ";
clear: both;
height: 0;
}


* html #menu            { zoom: 1; } /* IE6 */
[[Image: スライド15.jpg |340px]]
*:first-child+html #menu { zoom: 1; } /* IE7 */
[[Image: スライド16.jpg |340px]]
<youtube width="450" align="left">XMiheA1sWOA</youtube>
{{-}}


<h3>Potential Barrier</h3>
[[Image: Potential_energy.png|340px]]
Our Gate is made of DNA, so it has negative electric charge. Single stranded DNA has negative electric charge, too. Here is a graph at potential energy around the tube. GATE size means the length of the Gate. If the potential energy is high, it is difficult for single stranded DNAs to enter the Gate. If the radius of the Gate is 1.5 times larger than now design, potential energy decreases and to enter the Gate is easier.
[http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation You can see details in simulation page ]
{{-}}


/* 目次 */
div#mokuji {width: 1000px;
margin-left: auto;
margin-right: auto;
background-color: #f5f5dc}


div#mokuji h2 { background-color: # f5f5dc;
<h2>Porter</h2>
font-size: 1.50em;
<h3>Principle</h3>
color: #000000;
[[Image:スライドGif.gif|left|400px]]
line-height: 45px;
In the concept of Cell Gate, there are two problems. for making CELL-GATE.
padding-left: 12px;
<ul>How to pull the target DNA into GATE ?</ul>
margin-bottom: 0}
<ul>How to pass the target through GATE ?</ul>


div#mokuji h3 {border-bottom:solid 3px #66aa66;
To solve these problems, we propose a nano-system made of ssDNAs called "Porter". Porter stands in line inside the GATE, selectively "pull" the target DNA.
font-size: 1.50em;
line-height: 22px;
padding-left: 12px;
margin-top: 30px;
margin-bottom: 0;
clear: both}


This idea is supported by [http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation GATE simulation], which shows that target DNA can not enter GATE by itself. So, the work of PORTER is to pull and bring the target DNA inside GATE.


ol#mokuji {font-size: 1.00em;
We designed PORTER having some loop structures when it hybridizes with the target. So when the target attaches to Porter, Porter shrinks, or in other words it pulls the target DNA into the Gate. As a result the target enter GATE.
margin-left: 0;
padding-left: 0}


The inner Porter has longer complementary sequences to the target and thus  higher bonding energy than from the one at the entrance of the Gate(Porter1). This design enables the target to move to the inner Porter(Porter2 and Porter3). In experiment, we designed and used the sequences below.


/* 本文 */
{{-}}
<center>
[[Image: 配列.png|center|500px]]
div#Content {width: 1000px;
{{-}}
margin-left: auto;
margin-right: auto;
      }


div#Content h2 { background-color: # f5f5dc;
'''Blue''':Target '''Red''':This part is complementary with target  '''Green''':Spacer
font-size: 1.50em;
color: #000000;
line-height: 10px;
padding-left: 12px;
margin-top: 20px;
margin-bottom: 0}




div#Content h3 {background-color: #ffffff;
※Porter3 use only electrophoresis.
background-image: url(maru-skyblue.png);
----
background-repeat: no-repeat;
[[Image: スクリーンショット 2012-10-28 8.38.23.png|center|400px]]
background-position: 0px 2px;
We should note these described above are the sequences for electrophoresis experiments. Additional sequences to attach the GATE are included in the designed Porter sequences of the GATE.
font-size: 0.875em;
line-height: 22px;
padding-left: 26px;
margin-top: 30px;
margin-bottom: 0;
margin-left: 12px;
margin-right: 12px}


div#Content p {font-size: 1.25em;
line-height: 1.6;
margin-top: 10px;
margin-left: 12px;
margin-right: 12px}
</center>
</style>
</head>


<body>
{{-}}
<div id="Container">
   
<ul id="menu">
<li><a href="http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai">Top</a></li>
<li>
<a href="#">Project</a>
<ul>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea">Idea</a>
</li>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation">Simulation</a>
</li>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Design">Design</a>
</li>
</ul>
</li>
<li>
<a href="#">Experiment</a>
<ul>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result">Result</a>
<ul>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Porter">Porter</a>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result#Cylinder">Cylinder</a> </li>
<li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Result# Vesicle">Vesicle</a>
</li>
</ul>
<li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Method">Method</a> </li>
</ul>
</li>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Diary">Diary</a>
</li>
<li>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Team ">Team</a>
</li>
</ul>


<!-- コンテンツ -->
<h3>Simulation</h3>
<div id="Content">
Coarse grained simulation in which one nucleotide is assumed as one bead indicates that long Porter can bind to the target, but toehold structure of the same affinity cannot catch the target.<br>
<!--
<table border="0" align="left" vertical-align: middle;>
<a name="Motivation"></a><h2>Motivation</h2>
<p>
現在チャネルの研究は進められているが、それらは単純な穴であり、選択性を持って物質交換を行うものは少ない。そこで我々は選択性をもったチャネルの開発に着手した。</br>
我々が設計したチャネルはこうである。まず、穴としてDNAオリガミで作った六角柱を用意（Gate）。その内側に一本鎖DNAを数本並べておく。この一本鎖DNAをSelectorと名付けた。DNAはその相補性により、DNAはもとよりRNAやタンパク質などの様々な生体分子と相互作用を持ち、また簡単に配列を変更することが可能であるため、六角柱内に一本鎖DNAが並ぶ構造によって、汎用性を持った選択的なチャネルを作成することが可能である。Selectorの配列はチャネルの奥に行くほどターゲット分子との結合を強いように設計する。こうすることでターゲット分子は選別されながら、チャネルの出口へ向かって行くことができる。</br>
また、我々はチャネルの取り付く細胞膜のモデルとしてリポソームを使う。
</p>
-->
<a name="ProjectPlan"></a><h2>Project plan</h2>
<p>
Our project is divided large three parts, Selector, Gate and Liposome.</br>
So we'll do each experiments abreast and finally we'll mix. </br>
 
我々のプロジェクトは大きく三つに分けることができる。セレクター、チャネル、リポソームである。そこで我々はこの三つの実験を並行して行う。それぞれの実験がうまくいったところで組み合わせる。（D-Hertを分解した画像をこの下に欲しい）
</p>
 
<a name="Selector"></a><h2>Selector</h2>
<p>
Working Selector</br>
<div align="center">
<img src="http://openwetware.org/images/4/45/Suceed.gif" width="420px" height="300px"><br>
</div>
  Inside the gate, a cascade of three single stranded DNAs is planted. We named the DNAs Selector1, Selector2, and Selector3 from the outside of the gate. In addition, another Selector, which is called Selector4, is in the liposome.<br>
[In the gate]  Selector1and 2 have complementary sequences to a target oligonucleotide here and there and consecutive adenine sequences in other portion. We made an attempt to capture a target distant from the gate with high specificity. So we lay out a long Selector1. By catching a target and making loops, it can shrink and go in the gate.<br>
  Selector3 is complementary to the target, but it is shorter than the target. When it binds to the target, the upper end of the target makes a toehold.<br>
  We designed the inner Selector has higher bonding energy. So once the outer-most ssDNA binds to a target, the target is passed to the inner-ones one by one.<br>
[In liposome]  Selector4 is perfectly complementary to the target. After the target reaches Selector3, Selector4 conveys the target into liposome.<br>
<br>
<table style="clear:right;width:650px;border-style:solid;border-width:2px;margin:0 auto">
<tr>
<th style="width:100px;">Name</th><th style="width:450px;">Sequence(5' to 3')</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;">* - ACTAG<font color="green">TGAG</font><font color="orange">TGCAGCAGTCGTACCA</font></td><td style="border-style:solid;border-width:1px;"></td>
</tr>
<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;">30.6</td>
<td>
<youtube width="450" align="left">NXo4cYKkrF0&border=1&color1=0x6699&color2=0x54abd6</youtube>
<html><div style="clear:both;"></div></html>
</td>
<td width="20">
</td>
<td>
<youtube width="450" align="left">qidZS1pI0lc&border=1&color1=0x6699&color2=0x54abd6</youtube>
<html><div style="clear:both;"></div></html>
</td>
</tr>
</tr>
<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;">36.5</td>
<td align="center">
Porter can binds to the target
</td>
<td width="20">
</td>
<td align="center">
Toehold structure cannot bind to the target
</td>
</tr>
</tr>
<tr>
</table><br>
<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>
[http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation See detail in simulation page]
<tr>
<td style="border-style:solid;border-width:1px;">Selector4</td><td style="border-style:solid;border-width:1px;"><font color="red">TGGTACGACTGCTGCA<font color="blue">CTCA</font></font></td><td style="border-style:solid;border-width:1px;">68.0</td>
</tr>
</table>
<br>
*Red-orange and blue-green regions are complementary DNA sequences.<br>
*Black region is added to differ the molecular weight of each sample(for distinguishing them during electrophoresis).<br>
<br>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Selector">Experiment page</a>
 
</p>
 
 
 
<!--Gateコンテンツ-->
 
 
 
 
<a name="Gate"></a><h2>Gate</h2>
<font size="4">
<a name="Strategy"></a><h3>Strategy</h3>
</font>
<p>
 
 
Gate should be able to transport the target with selector inside gate and go through cell membrane.
To transport the target with selector, we decided to make hexagonal tube as gate.
The reasons we adopted hexagonal tube as gate are that surfaces of hexagonal tube are suitable for being attached selector,
high strength of honeycomb structure are easy to be observed. To go through cell membrane, we placed the staple attached lipid on center of gate.</br>
We expect gate is introduced liposome simultaneously with creation of liposome.
In addition, we attached edge of the gate to adenine staple like a "mustache" to make easy watching by AFM and interrupt other DNA approaching.
We think because of our selector 1 is enough long, only target is transported into the gate. </br>
In addition to this, we made the cholesterol hexagonal tube. The reason that designed this tube is coupling into a liposome film using a characteristic of the cholesterol like a lipid. (cf.Figure 2.3 )
</br>
<font size="4">
<h3>Structure image</h3>
</font>
<p>
 
This is the hexagonal tube design by caDNAno using honeycomb structure.</br>
<br>
 
<img src="http://openwetware.org/images/9/91/Cadnano3D.gif" width="315px" height="405px" alt="Structure image"/>
</br>
<p>
We made 3shape’s hexagonal tube.</br>
1: Mere hexagonal tube</br>
2: Hexagonal tube with the adenine at the entrance</br>
3: The cholesterol hexagonal tube</br>
 
<img src="http://openwetware.org/images/c/c4/スクリーンショット_2012-10-15_1.11.25.png" width="474px" height="351px"/ >
 
<img src="http://openwetware.org/images/0/0b/スクリーンショット_2012-10-15_1.11.35.png" width="471px" height="356px" align="right"/ >
<img src="http://openwetware.org/images/5/5e/スクリーンショット_2012-10-15_1.22.38.png" width="456px" height="351px"/ >
</br>
 
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Gate">
</br>
Experiment page</a>
</font>
</p>
 
 
</br>
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 




<a name="Membrane"></a><h2>Membrane</h2>
<h2>Membrane: How to implement the GATE</h2>
<p>
<h3>Cell model</h3>
We use liposomes as a model of cell membrane. To insert cell-gate into the</br>
To insert the Gate in cell membranes is essential for the CELL GATE. We used artificial lipid membrane, liposomes, as model cell membranes, to test implementation of our CELL GATE into membrane. As a preliminary step to insertion of the GATE into the liposome, we designed a smaller Gate named Mini-gate. We attempted to insert the Gate and Mini-gate into liposomes and we confirmed they inserted into liposomes by fluorescence microscopy or by SPR analysis.
liposome, we stretched out ssDNA of 10 nt from the side of the hexagonal tube. Then,</br>
<h3>Cholesterol-leg</h3>
we extend the complementary ssDNA, and modified the cholesterol at the end of them.</br>
To implement the Gate in membranes, we attached single-stranded DNA of 10 bases at the middle point of the GATE outside surface. A hydrophobic molecule, Cholesterol, was conjugated into the complementary DNA of the attached DNA. We expected that the GATE with cholesterol legs can be implemented into the hydrophobic portion of the liposome.<br>
We choose the cholesterol because cholesterol is strongly hydrophobic. We expect</br>
There is a possibility that the GATE with cholesterol legs lie on the membrane surface, and is not inserted. Thus, installing a module for insertion was required. For the aim, we designed that the Mini-gate remains a large amount of single stranded region of M13. We expected that this single stranded region of M13 breaks electrostatic symmetry of the Mini-Gate, and enables to stand vertically to penetrate the membrane by repulsion.
that cholesterol penetrate into the hydrophobic portion of the liposome. We confirmed</br>
{{-}}
the tube modifying the cholesterol by electrophoresis. We use fluorescein to confirm</br>
[[Image: スクリーンショット_2012-10-15_1.11.35.png |300px]]
that the tube insert into the liposome correctly.</br>
[[Image: スクリーンショット_2012-10-15_1.22.38.png |300px]]
（細胞膜のモデルとして、リポソームを使用する。リポソームにセルゲートを刺さるように</br>
[[Image: みにげーと.png |300px]]
するために、六角形筒の横から10塩基のシングルストランドDNAを伸ばした。さらに、</br>
{{-}}
相補的なシングルストランドDNAを伸ばして、それらの末端にコレステロール修飾を行</br>
った。コレステロールを修飾したのは、コレステロールが強い疎水性であるからである。</br>
コレステロールがリポソームの疎水性部分に入り込むことで、筒がリポソームに刺さりや</br>
すくなると考えた。コレステロール修飾は電気泳動で確かめた。筒がリポソームに刺さ</br>
っているかを確かめる方法として、蛍光分子を利用する。）</br>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Membrane">Experiment page</a>
</p>
 
 
 
 
 
 
</div>
 
</body>
</html>

Latest revision as of 19:43, 27 October 2012

<html>

<head><style>#column-one { display:none; width:0px;} .container{background-color: #f5f5f5; margin-top:50px} .OWWNBcpCurrentDateFilled {display: none;}

  1. content {width: 0px; margin: 0 auto auto 0; padding: 1em 1em 1em 1em; align: center;}
  2. column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}

.firstHeading {display:none; width:0px;}

  1. globalWrapper{width:1280px; margin:auto}

body {background: #F0F0F0 !important;}

  1. column-one {display:none; width:0px;background-color: #f0f0f0;}
  2. content{border:none;margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: 800px;background-color: #f0f0f0; }

.container{ width:1280px; margin: auto; background-color: #f0f0f0; text-align:justify; font-family: helvetica, arial, sans-serif; color:#f0f0f0; margin-top:25px; }

  1. bodyContent{ width: 1267px; align: center; background-color: #f0f0f0;}
  2. column-content{width: 1280px;background-color: #f0f0f0;}

.firstHeading { display:none;width:0px;background-color: #f0f0f0;}

  1. header{position: center; width: 800px;background-color: #f0f0f0;}
  2. footer{position: center; width:1280px;}

</style><header>

</html>



<html> <head> 

   <title>Team Sendai Top</title>
   <script type="text/javascript" src="http://code.jquery.com/jquery-latest.min.js"></script>
   <script type="text/javascript">
       $(function() {
         if ($.browser.msie && $.browser.version.substr(0,1)<7)
         {

$('li').has('ul').mouseover(function(){ $(this).children('ul').show(); }).mouseout(function(){ $(this).children('ul').hide(); }) 

         }
       });        
   </script>

<style>

body{ background-image:url(http://openwetware.org/images/7/78/Office.png); /*壁色と壁紙設定*/ background-repeat:repeat;/*繰り返さない場合はno-repeatに変更*/ font:"メイリオ", "ＭＳ Ｐゴシック", Osaka, "ヒラギノ角ゴ Pro W3"; color: #333333; margin:0px; padding:0px; }

  1. Container

{ Background-image: url(http://openwetware.org/images/6/62/Wall4.png); width: 960px; margin: auto; }

/* Main menu */

  1. menu

{ width: 100%; margin: 0; padding: 10px 0 0 0; list-style: none; background: #111; background: -moz-linear-gradient(#444, #111); 

   background: -webkit-gradient(linear,left bottom,left top,color-stop(0, #111),color-stop(1, #444));

background: -webkit-linear-gradient(#444, #111); background: -o-linear-gradient(#444, #111); background: -ms-linear-gradient(#444, #111); background: linear-gradient(#444, #111); -moz-border-radius: 50px; border-radius: 50px; -moz-box-shadow: 0 2px 1px #9c9c9c; -webkit-box-shadow: 0 2px 1px #9c9c9c; box-shadow: 0 2px 1px #9c9c9c; }

  1. menu li

{ float: left; padding: 0 0 10px 0; position: relative; line-height: 0; }

  1. menu a

{ float: left; height: 25px; padding: 0 25px; color: #999; text-transform: uppercase; font: bold 12px/25px Arial, Helvetica; text-decoration: none; text-shadow: 0 1px 0 #000; }

  1. menu li:hover > a

{ color: #fafafa; }

  • html #menu li a:hover /* IE6 */

{ color: #fafafa; }

  1. menu li:hover > ul

{ display: block; }

/* Sub-menu */

  1. menu ul

{ 

   list-style: none;
   margin: 0;
   padding: 0;    
   display: none;
   position: absolute;
   top: 35px;
   left: 0;
   z-index: 99999;    
   background: #444;
   background: -moz-linear-gradient(#444, #111);
   background: -webkit-gradient(linear,left bottom,left top,color-stop(0, #111),color-stop(1, #444));
   background: -webkit-linear-gradient(#444, #111);    
   background: -o-linear-gradient(#444, #111);	
   background: -ms-linear-gradient(#444, #111);	
   background: linear-gradient(#444, #111);
   -moz-box-shadow: 0 0 2px rgba(255,255,255,.5);
   -webkit-box-shadow: 0 0 2px rgba(255,255,255,.5);
   box-shadow: 0 0 2px rgba(255,255,255,.5);	
   -moz-border-radius: 5px;
   border-radius: 5px;

}

  1. menu ul ul

{ 

 top: 0;
 left: 150px;

}

  1. menu ul li

{ 

   float: none;
   margin: 0;
   padding: 0;
   display: block;  
   -moz-box-shadow: 0 1px 0 #111111, 0 2px 0 #777777;
   -webkit-box-shadow: 0 1px 0 #111111, 0 2px 0 #777777;
   box-shadow: 0 1px 0 #111111, 0 2px 0 #777777;

}

  1. menu ul li:last-child

{ 

   -moz-box-shadow: none;
   -webkit-box-shadow: none;
   box-shadow: none;

}

  1. menu ul a

{ 

   padding: 10px;

height: 10px; width: 130px; height: auto; 

   line-height: 1;
   display: block;
   white-space: nowrap;
   float: none;

text-transform: none; }

  • html #menu ul a /* IE6 */

{ height: 10px; }

  • first-child+html #menu ul a /* IE7 */

{ height: 10px; }

  1. menu ul a:hover

{ 

   background: #0186ba;

background: -moz-linear-gradient(#04acec, #0186ba); background: -webkit-gradient(linear, left top, left bottom, from(#04acec), to(#0186ba)); background: -webkit-linear-gradient(#04acec, #0186ba); background: -o-linear-gradient(#04acec, #0186ba); background: -ms-linear-gradient(#04acec, #0186ba); background: linear-gradient(#04acec, #0186ba); }

  1. menu ul li:first-child > a

{ 

   -moz-border-radius: 5px 5px 0 0;
   border-radius: 5px 5px 0 0;

}

  1. menu ul li:first-child > a:after

{ 

   content: '';
   position: absolute;
   left: 30px;
   top: -8px;
   width: 0;
   height: 0;
   border-left: 5px solid transparent;
   border-right: 5px solid transparent;
   border-bottom: 8px solid #444;

}

  1. menu ul ul li:first-child a:after

{ 

   left: -8px;
   top: 12px;
   width: 0;
   height: 0;
   border-left: 0;	
   border-bottom: 5px solid transparent;
   border-top: 5px solid transparent;
   border-right: 8px solid #444;

}

  1. menu ul li:first-child a:hover:after

{ 

   border-bottom-color: #04acec;

}

  1. menu ul ul li:first-child a:hover:after

{ 

   border-right-color: #04acec; 
   border-bottom-color: transparent;

}


  1. menu ul li:last-child > a

{ 

   -moz-border-radius: 0 0 5px 5px;
   border-radius: 0 0 5px 5px;

}

/* Clear floated elements */

  1. menu:after

{ visibility: hidden; display: block; font-size: 0; content: " "; clear: both; height: 0; }

  • html #menu { zoom: 1; } /* IE6 */
    first-child+html #menu { zoom: 1; } /* IE7 */

/* 目次 */ div#mokuji {width: 960px; margin-left: auto; margin-right: auto; background-color: #f5f5dc}

div#mokuji h2 { background-color: # f5f5dc; font-size: 1.50em; color: #000000; line-height: 45px; padding-left: 12px; margin-bottom: 0}

div#mokuji h3 {border-bottom:solid 3px #66aa66; font-size: 1.50em; line-height: 22px; padding-left: 12px; margin-top: 30px; margin-bottom: 0; clear: both}


ol#mokuji {font-size: 1.00em; margin-left: 0; padding-left: 0}


/*文字の設定*/ h1{ font-family: 'Times New Roman', 'Times', serif; font-size: 2.00em; letter-spacing: 2px; padding-left:10px; margin: 0px; }


h2{ font-family: 'Times New Roman', 'Times', serif; 

     	font-size:1.8em;

border-left: 10px solid #000066; 

         	border-bottom:solid 1px #000099;/*文字の下に線を入れる設定*/
         	width:900px;

padding-left: 5px; color:#333333; margin-top: 15px; margin-bottom: 5px; }

h3{ font-family: 'Times New Roman', 'Times', serif; 

     font-size:1.60em;
       color:# 333333;

}

p{ 

   	font-family: 'Times New Roman', 'Times', serif  	font-size:1.40em;/*全体の文字サイズ*/

line-height:150%;/*全体で使う、文字と文字の行間*/ 

         	margin-left:5px;

}

p img{ 

    	float:left;
        	margin-top:5px;  /*写真の左にスペースを空ける*/

margin-left:5px; /*写真の左にスペースを空ける*/ margin-right:10px; /:写真と文字の間隔*/ }


h5{ 

     font-size:1.60em;

border: solid 1px #111111; 

       color:#ffffff;

background-color:#FFA500; line-height: 30px; padding-left:10px; margin-top: 10px; margin-bottom: 1px; }

h6{ 

     font-size:120%;

border: solid 1px #111111; 

       color:#ffffff;

background-color:#006400; line-height: 30px; padding-left:10px; margin-top: 10px; margin-bottom: 1px; }

  1. red{
     font-size:120%;

border: solid 1px #111111; 

       color:#ffffff;

background-color:#DC143C; line-height: 40px; padding-left:10px; margin-top: 10px; margin-bottom: 1px; }

  1. blue{
     font-size:120%;

border: solid 1px #111111; 

       color:#ffffff;

background-color:#191970; line-height: 40px; padding-left:10px; margin-top: 10px; margin-bottom: 1px; }

  1. pop {

width: 50%; margin: 0px; float: left; }

  1. pop h2 {

width: 90%; margin: 0px; float: left; }

  1. pop p {

width: 90%; margin: 0px; float: left; }


/*topに戻る*/

  1. ttop {position:fixed;bottom:140px;left:auto;margin:0 0 0 960px; /* マージン：上 右 下 左 */width:41px;height:390px;background:url(http://openwetware.org/images/e/ed/Cellgate.gif) no-repeat left bottom;}/* IE6以下用 */* html #ttop {margin:0 0 -390px 0;position:relative;bottom:490px; /* 上で設定した ttopの高さ390px＋下100px */left:960px;}#ttop:hover {background:url(http://openwetware.org/images/a/a7/029.png) no-repeat left bottom;/* 画像の高さによって適当に調整 */}a.page_top {display:block;width:41px;height:390px;}


</style> </head>


<body> <div id="Container"> <!-- Menu --> <ul id="menu"> <li><a href="http://openwetware.org/wiki/Biomod/2012/Tohoku/Team_Sendai ">Home</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea ">Project</a></li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Design">Design</a> </li> <li><a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation">Simulation</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Experiment ">Experiment</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Future Application">Future Application</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Diary">Diary</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Team ">Team</a> </li> <li> <a href=" http://openwetware.org/wiki/Biomod/2012/Tohoku/Team Sendai/header"></a> </li>

</ul>

<div id="ttop"> <a href="#top" class="page_top" onfocus="this.blur();" onclick="scrollTo(0,0); return false;" title="Top"></a></div>

</body> </div> </html>


Design

Gate

Size / Structure

What structure is most suitable for the Gate? The Gate has to connect inside and outside of the cell. So we decided to apply a hexagonal tube nanostructure made of DNA origami. We refer "A logic-gated nanorobot for targeted transport of molecular payloads" (SM Douglas, I Bachelet, GM Church - Science Signalling, 2012) for the hexagonal tube structure of DNA origami.
Next, we made a simulation in order to examine the size of the structure. The size of the tube must be small enough not to pass freely through anything. However, it must be large enough to pass through the desired product. The gate which made of DNA origami has negative electric charge. So if the gate is too small, target can't enter the Gate. According to simulation, our Gate size determined 24*24*33nm. This size is suitable to transport the target.

DNA origami

We used caDNAno to design the hexagonal tube structure. This Gate tube is made from 6792bp M13mp18 and a lot of single stranded DNAs. And the Gate has double hexagonal structure because I think that is stronger than single hexagonal structure.


Potential Barrier

Our Gate is made of DNA, so it has negative electric charge. Single stranded DNA has negative electric charge, too. Here is a graph at potential energy around the tube. GATE size means the length of the Gate. If the potential energy is high, it is difficult for single stranded DNAs to enter the Gate. If the radius of the Gate is 1.5 times larger than now design, potential energy decreases and to enter the Gate is easier. You can see details in simulation page


Porter

Principle

In the concept of Cell Gate, there are two problems. for making CELL-GATE.

    How to pull the target DNA into GATE ?
    How to pass the target through GATE ?

To solve these problems, we propose a nano-system made of ssDNAs called "Porter". Porter stands in line inside the GATE, selectively "pull" the target DNA.

This idea is supported by GATE simulation, which shows that target DNA can not enter GATE by itself. So, the work of PORTER is to pull and bring the target DNA inside GATE.

We designed PORTER having some loop structures when it hybridizes with the target. So when the target attaches to Porter, Porter shrinks, or in other words it pulls the target DNA into the Gate. As a result the target enter GATE.

The inner Porter has longer complementary sequences to the target and thus higher bonding energy than from the one at the entrance of the Gate(Porter1). This design enables the target to move to the inner Porter(Porter2 and Porter3). In experiment, we designed and used the sequences below.



Blue:Target Red:This part is complementary with target Green:Spacer


※Porter3 use only electrophoresis.

We should note these described above are the sequences for electrophoresis experiments. Additional sequences to attach the GATE are included in the designed Porter sequences of the GATE.



Simulation

Coarse grained simulation in which one nucleotide is assumed as one bead indicates that long Porter can bind to the target, but toehold structure of the same affinity cannot catch the target.

EmbedVideo received the bad id "NXo4cYKkrF0&border=1&color1=0x6699&color2=0x54abd6" for the service "youtube".

<html><div style="clear:both;"></div></html>

EmbedVideo received the bad id "qidZS1pI0lc&border=1&color1=0x6699&color2=0x54abd6" for the service "youtube".

<html><div style="clear:both;"></div></html>

Porter can binds to the target

Toehold structure cannot bind to the target



See detail in simulation page


Membrane: How to implement the GATE

Cell model

To insert the Gate in cell membranes is essential for the CELL GATE. We used artificial lipid membrane, liposomes, as model cell membranes, to test implementation of our CELL GATE into membrane. As a preliminary step to insertion of the GATE into the liposome, we designed a smaller Gate named Mini-gate. We attempted to insert the Gate and Mini-gate into liposomes and we confirmed they inserted into liposomes by fluorescence microscopy or by SPR analysis.

Cholesterol-leg

To implement the Gate in membranes, we attached single-stranded DNA of 10 bases at the middle point of the GATE outside surface. A hydrophobic molecule, Cholesterol, was conjugated into the complementary DNA of the attached DNA. We expected that the GATE with cholesterol legs can be implemented into the hydrophobic portion of the liposome.
There is a possibility that the GATE with cholesterol legs lie on the membrane surface, and is not inserted. Thus, installing a module for insertion was required. For the aim, we designed that the Mini-gate remains a large amount of single stranded region of M13. We expected that this single stranded region of M13 breaks electrostatic symmetry of the Mini-Gate, and enables to stand vertically to penetrate the membrane by repulsion.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2012/TeamSendai/Design&oldid=647671"