Biomod/2012/TeamSendai/Design: Difference between revisions

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<h1>Cell Gateの構造</h1>
<p>
Cell Gateの全体像を紹介。下の図をさらにシンプルで現実に即したものにする感じで。（Membraneにコレステロールの脚のついた六角柱のGateが貫通して、さらにGateが断面図になっていて中のPorterが見えるような図で説明）。Gate, Porter, Membraneが何の役割をしているのかさらっと説明。
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<h1>Gate</h1>
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<p>
なぜ六角柱にしたのかとか、DNAオリガミである理由とかが言えたらいいと思うけど、あるのでしょうか…？　ショーンの論文を参考にしましたと画像付きで説明。caDNAnoのデータは…載せる？
</br>
</br>
仮の文章↓
<h2>Determining the structure of the Gate</h2>
<img src=" http://openwetware.org/images/f/f1/Shawnpicture.png" align="right" width="400px" height="400px">
What structure is most suitable to the Gate?</br>
Gate have to connect inside to outside of the cell.</br>
So Gate have to be the structure like a tube.</br>
And it is desireble that is easy to make.</br>
We decided to use the structure of the hexagonal tube.</br>
The reason is because we think there is a reference on the hexagonal structure of DNA origami（ショーンの論文を紹介）,
and can take advantage of that knowledge.</br>
                                                                                                       
</br>


<h2>Gate size</h2>
Next, We made a simulation in order to examine the size of the structure.
Must not the size that it is the tube through anything.
It must be large enough to be passed through the desired product, however.
The gate made of DNA origami have negative electric charge.
So if the gate is too small, target can't enter the Gate. 
According to simulation, our Gate size determined 24*33*33nm.(普通の筒の画像載せる)
This size is suitable totransport the target.


</br>


<h2>Penetrate the mambrane</h2>
We also have the aim of this GATE stab in the cell membrane,
it can not sting in the cell membrane of normal hexagonal tube.However, We can create a tube with a different structure by exchanging some staple.
We have designed a simple tube first,and I have to be attached anywhere in the structure to replace the staple .
We can later add functionality to an existing structure using this method.We thought that to have an affinity for lipid membrane with the DNA
that can be modified cholesterol on the side of the tube by this method.In addition, we have also designed DNA-like beard at the entrance of the tube.
We expect the effect of electrostatically repel force with DNA which are not intended.Our tube is small,
so we designed the tube to connect to each other and be long in order to easily confirmed using AFM.
By replacing the staple, this structure is also removably.(その他の筒の絵)
</br>


</p>
{{:Biomod/2012/Tohoku/Team_Sendai/header}}


<h1>Porter</h1>
<p>
どのような種類のPorterを、どのような理由でそこに配置したのかとか…？（入口のPorterはシミュレーションから長くなくてはならないとか…？）Selectorの配列を表示。</br>
仮の文章↓</br>


<h2>What’s “Porter”?</h2>
<div id="Container">
When we consider making Cell Gate, there are two problems.
   
One is how to pull the target oligonucleotide into Cylinder, and the other is how to pass the target.
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To solve these problems, we propose a nano structure of ssDNA and named it “Porter”.
<div id="Content">
Porter stands in line in Cylinder, pulls the target, and transports it.
</br>


<h2>Electric repulsion</h2>
<h1>Design</h1>
We did simulation to find the best length and place of Porter.  
<h2>Gate</h2>
The simulation shows that the target, which is negatively charged, can approach to Cylinder, only when there’s a certain distance between them.  
<h3>Size / Structure</h3>
To enable the target to go enough close to Cylinder, Porter has to be longer than the distance and carry the target inside Cylinder.
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>
</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>
{{-}}


<h2>Loop structure</h2>
<h3>DNA origami</h3>
We designed Porter having some loop structures when it hybridizes with the target.
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.
  Porter has some complementary sequences to the target here and there.
So after the target attaches to Porter at the end of Porter, Porter shrinks and pulls the target.
Finally the target passes the distance otherwise unable to go through.
</br>


<h2>Transportation of the target</h2>
[[Image: スライド15.jpg |340px]]
The inner Porter has more complementary sequences to the target and has higher bonding energy from the entrance of Cylinder.  
[[Image: スライド16.jpg |340px]]
This design enables the target to move to the inner Porter because of the combination stability.
<youtube width="450" align="left">XMiheA1sWOA</youtube>
</br>
{{-}}


<h2>Experiment</h2>
<h3>Potential Barrier</h3>
In experiment, we apply the sequences below.  
[[Image: Potential_energy.png|340px]]
We compared the effectiveness and efficiency of Porter with that of toehold structure.
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.
<DNA sequences>(実際の配列をお願いします)
[http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation You can see details in simulation page ]
These are the sequences for electrophoresis, and only a part of the actual Porter sequence. These are the extracted region binding to the target.
{{-}}
When planted in the gate, Porter has more spacer sequences of 10base at the root in addition to the sequences above,
to reduce the Coulomb's force produced by the wall of Cylinder.
</br>


</p>


<h1>Membrane</h1>
<h2>Porter</h2>
<p>
<h3>Principle</h3>
どの脂質をどういう理由で選び、どのように配合したのかとか…</br>
[[Image:スライドGif.gif|left|400px]]
仮の文章↓</br>
In the concept of Cell Gate, there are two problems. for making CELL-GATE.
<h2>Insert the mini-gate</h2>
<ul>How to pull the target DNA into GATE ?</ul>
We use liposomes as a model of cell membrane. First, we designed the composition of
<ul>How to pass the target through GATE ?</ul>
liposome and made the liposomes. And then, we prepared a preliminary step to insert
cell gate into the liposome. We designed a smaller tube named “mini-gate” as a model of
cell-gate. We attempted to insert it into our liposomes and we observed how “mini-gate”
inserted into liposomes.
</br>


<h2>Modification of mini-gate</h2>
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.
Similar to the actual cell gate, we also stretched single-stranded DNA of 10 bases that
can be modified cholesterol from the side of “mini-gate” as Gate team did. We attached
cholesterol to the side of the “mini-gate” using that single-stranded DNA, and expected that
these enter into the hydrophobic portion of the liposome. Then, it is likely to that the tube
stick to the liposome.
</br>


<h2>Penetrate with standing</h2>
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.
However, we modified cholesterol to stick to the liposome, it is considered that the “mini-
gate” would be in a state of lying on the liposome. Therefore, when we designed the mini-
gate, we made one side too much for the extra M13 deliberately. We expect that the
electrostatic symmetry of the tube is broken by this extra M13, and the tube penetrate the
membrane standing vertically by repulsion.
</br>


</p>
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.
</br>
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</br>
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</br>
</br>


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.


{{-}}
[[Image: 配列.png|center|500px]]
{{-}}


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




※Porter3 use only electrophoresis.
----
[[Image: スクリーンショット 2012-10-28 8.38.23.png|center|400px]]
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.




<p>ここから下、昔の文章なので参考までに…</p>
{{-}}
<a name="Porter"></a><h2>Porter</h2>
<img src="http://openwetware.org/images/4/45/Suceed.gif" align="right" width="210px" height="150px">
<p>
Tubeの中に一列に並び、チャネルの原動力となる一本鎖DNAの列をPorterと名付ける。Porterは入口から奥へ行くほどターゲット分子との相補的な配列が多くなるように設計する。こうすることで“意図的に”エネルギー勾配を作り出すことができ、物質を移動させることができるようになる。</br>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Idea">Idea</a>で言ったように、物質の通り道となるチャネルの外壁はDNAオリガミで作るため、短い一本鎖DNAではDNAのカスケードの流れにターゲット分子を乗せることはできない。Reference: <a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation">Simulation</a>　＜/br＞
また、かといってただ単純に一本鎖DNAを伸ばしただけでは、クーロン力の壁を越えられず、先端の相補的な配列にターゲット分子が結合するのみで、一本鎖DNAは伸びきったまま、Tubeの中に物質を取り込むことはできないだろう。（画像載せる）</br>
そこでPorterがターゲット分子と結合した場合、ループ構造をとるように設計した。Porterの各所にある相補的な配列に、段階的にターゲット分子が結合することにより、物体を引き寄せ、Tubeの中に物質を引き入れることができるようになるだろう。（画像載せる）また捕集できる場所が多くなるため捕集率も向上すると考えられる。（画像載せる）</br>
実験では以下の配列を用いて、トーホールドとループ構造の比較を行った。（配列載せる）</br>
また、これは電気泳動用の、ターゲットとの結合に関係する部分だけを抜き出した配列である。実際のTubeに搭載する場合はこの配列の他に、根元から10bpほどスペーサー配列を組み込むことで、移動中に壁から受けるクーロン力の影響を減らしている。</br>
実験では主に、Tubeの中にターゲットを引き込むことを主眼としているが、入口から入って行ったターゲットを最も出口に近いPorterまで移動させ出口から放出するには、最も出口に近いPorterとターゲットの関係をトーホールドにしておき、結合した状態でさらにターゲットに相補的配列を持つ一本鎖DNAを投入すればよいと考えられる。</br>
計算上、Tubeから出ることよりも、入ることのほうが難しい。</br>


( 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>
<h3>Simulation</h3>
[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>
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>
  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>
<table border="0" align="left" vertical-align: middle;>
  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">
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<th style="width:100px;">Name</th><th style="width:450px;">Sequence(5' to 3')</th><th style="width:100px;">Tm(°C)</th>
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<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>
<td>
<youtube width="450" align="left">NXo4cYKkrF0&border=1&color1=0x6699&color2=0x54abd6</youtube>
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</td>
<td width="20">
</td>
<td>
<youtube width="450" align="left">qidZS1pI0lc&border=1&color1=0x6699&color2=0x54abd6</youtube>
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</td>
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<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 align="center">
Porter can binds to the target
</td>
<td width="20">
</td>
<td align="center">
Toehold structure cannot bind to the target
</td>
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</table><br>
<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>
{{-}}
</tr>
[http://openwetware.org/wiki/Biomod/2012/TeamSendai/Simulation See detail in simulation page]
<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>
<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>




 
<h2>Membrane: How to implement the GATE</h2>
<!--Gateコンテンツ-->
<h3>Cell model</h3>
 
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.
 
<h3>Cholesterol-leg</h3>
 
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>
 
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.
<a name="Tube"></a><h2>Tube</h2>
{{-}}
<p>
[[Image: スクリーンショット_2012-10-15_1.11.35.png |300px]]
まず我々は円形断面のTubeを想定した。しかし、円形断面は力学的に見て強度が低い。そのためか、円形断面に関するDNAオリガミ構造体のデータも少なく、円形断面のTubeを作ることは難しそうであった。力学的に最も強度の高い構造は、三角形断面であるが、三角形断面は作成に必要な材料の数が多くなるなどの問題が挙げられる。また、三角形というある種特異的な断面を使用すると、目標物質の形状に関する汎用性が低下するおそれがある。断面はなるべく円に近いほうが良い。</br>
[[Image: スクリーンショット_2012-10-15_1.22.38.png |300px]]
そこで考えたのが六角形断面、ハニカム構造の断面を持つ六角柱型のTubeである。六角形は三角形を6つ並べた構造をしているため、比較的強度が高く、建築物の構造にも使われ、自然界では蜂の巣の中などにその形を見出すことが出来る。また、六角柱型のDNAオリガミ構造体に関する参考文献（ショーンの論文を紹介）もあり、その知見を利用することが出来る。よってTubeは六角柱で構成することにする。</br>
[[Image: みにげーと.png |300px]]
ただの六角柱では細胞膜に刺さることはできない。そこで、筒の側面にコレステロールを修飾したDNAを生やし脂質膜と親和性を持たせる。さらにそのままでは、脂質の上に寝た状態でいることが安定になるといけないので、六角柱のDNAオリガミを作製する際、わざと片側にM13を余らせることにした。このことで対称性が崩れ、反発力によって、Tubeが立ったまま細胞膜を貫通することができる。
{{-}}
 
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>
<h3>Structure image</h3>
<p>
 
This is the hexagonal tube design by caDNAno using honeycomb structure.（動いていない画像のほうがいいと思う）</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>
</p>
 
 
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<a name="Membrane"></a><h2>Membrane</h2>
<p>
We use liposomes as a model of cell membrane. To insert cell-gate into the　liposome, we stretched out ssDNA of 10 nt from the side of the hexagonal tube. Then,　we extend the complementary ssDNA, and modified the cholesterol at the end of them.　We choose the cholesterol because cholesterol is strongly hydrophobic. We expect　that cholesterol penetrate into the hydrophobic portion of the liposome. We confirmed　the tube modifying the cholesterol by electrophoresis. We use fluorescein to confirm　that the tube insert into the liposome correctly.
（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、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。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>
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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.

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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.

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