Biomod/2014/Kashiwa/Experiments: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
 
(233 intermediate revisions by the same user not shown)
Line 6: Line 6:
<style>
<style>


div#subContents {
div.imagebox {
    width: 200px;
  border: 1.7px dashed #2E2EFE; /* 1.枠線 */
    background: #FFFFFF;   
  background-color: #F0F8FF;  /* 2.背景色 */
    position: fixed;
  width: 310px; /* 横幅 */
    top: 100px;
  float: right; /* 右に配置 */
    left: 15px;
  margin: 0px 30px 0px 10px;
    z-index: 20;  
}
}   
p.image {
  text-align: center;
  width: 300px;
  height: 213px;
  margin: 5px;
}
p.caption {
  margin: 5px 10px;
  font-size: 90%; /* 5.文字サイズ */
  color: black; /* 6.文字色 */
}


* html div#subContents    {   
div.centerbox {
    position: absolute;   
   border: 1.7px dashed #2E2EFE; /* 1.枠線 */
    top: expression(eval(document.documentElement.scrollTop+100));   
   background-color: #F0F8FF; /* 2.背景色 */
} 
   width: 310px; /* 横幅 */
 
   margin: 0px 40px;
.category {
   float: left;
   border-style: double;
}
  border-width: thick;
   border-color: #483D8B;
   font-size: 12px;
   list-style-type:none;
   list-style-image:none;
  padding: 10px 3px 15px 8px;
  margin: 0px;  }


div.smallbox {
  border: 1.7px dashed #2E2EFE; /* 1.枠線 */
  background-color: #F0F8FF;  /* 2.背景色 */
  width: 210px; /* 横幅 */
  margin: 5px;
  float: right;
}
p.smallimage {
  text-align: center;
  width: 200px;
  height: 120px;
  margin: 5px;
}


h1.big{
h1.big{
   font-size :24px;
   font-size :16px;
  display: block;
   text-decoration: none;
   text-decoration: none;
   color: #000000;
   color: #000000;
　font-weight:bolder;
  margin: 10px 15px;
   margin: 25px 35px;
   font-weight: bolder;
   }
   }


h1.small{
h1.sub {
   font-size :20px;
   font-size:20px;
   display: block;
   font-weight: bolder;
  text-decoration: none;
   margin: 10px 5px;
   color: #000000;
}
　font-weight:bolder;
  }


div.name{
p.menu {
   font-size :16px;
   font-size :100%;
   color: #191970;
   line-height: 135%;
   font-weight:bolder;
   margin: 0px 40px;
  }
}


p.ex{
</style>
  font-size :100%;
  margin: 10px 65px;
  }


div.left img {
<script type="text/javascript"><!--
float: left;
function HideCBox( boxid ) {
position: relative;
  var target = document.getElementById(boxid);
margin: 0px 5px 0px 65px;
  if( target.style.display != "none" ) {
width: 300px;
      target.style.display = "none";
  }
  else {
      target.style.display = "";
  }
}
}
// --></script>


div.right img {
<style type="text/css"><!--
position: relative;
div.CollapsibleBox {
margin: 0px 65px 0px auto;
  /* ■ボックス全体■ */
width: 300px;
  border: 0px solid #4169E1; /* 枠線:濃青 */
float: right;
  font-size: 100%;            /* 文字サイズ:100% */
  width: 600px;              /* 横幅:600px */
}
div.CollapsibleBox div.CollapsibleBoxTitle {
  /* ■タイトル枠■ */
  background-color: #191970; /* 背景色:濃青 */
  color: white;               /* 文字色:白 */
  margin: 0px;
  padding: 0px;
  width: 600px;
}
div.CollapsibleBox div.CollapsibleBoxTitle p.cBoxTitle {
  /* ■タイトル文字列■ */
  font-size: 100% ;          /* 太字 */
  border: 1px solid #4B0082; /* 枠線:濃青 */
  margin: 0px;
  padding: 1px 0px;
}
div.CollapsibleBox div.CollapsibleBoxTitle p.cBoxButtons {
  /* ■ボタン群■ */
  margin: 0px;
  padding: 2px 10px;
  float: right;
  position: relative;
}
div.CollapsibleBox div.CollapsibleBoxTitle p.cBoxButtons a img {
  /* ■ボタンそのもの■ */
  border-width: 0px;    /* リンクの枠線を消す */
}
}
 
div.CollapsibleBox div.CollapsibleBoxBody {
div.tateleft img {
  /* ■本文枠■ */
float: left;
  margin: 0px;
margin: 0px 5px 0px 65px;
  padding: 2px 0px 2px 0px;
height: 300px;
  background-color: #ADD8E6; /* 背景色:淡青 */
  color: #191970; /* 文字色:暗青 */
}
}
 
div.CollapsibleBox div.CollapsibleBoxBody p {
div.tateright img {
  /* ■本文■ */
float: right;
  margin: 0px;
margin: 0px 65px 0px 5px:
  padding: 0.5em;   /* 内側の余白:0.5文字分 */
height: 300px;
}
}


</style>
</style>
<div id="subContents"> 
<ul class="category">
<li><a href="#1"><h3>Experiments</h3></a></li>
<li>The Receptor
<ul>
<li><a href="#1-1" title="CSS リファレンス一覧">Folding the Receptor</a></li> 
<li><a href="#1-2" title="Webデザインの一覧">Forming a Receptor dimer</a></li> 
<li><a href="#1-3" title="CSS ビギナー(初心者)向け一覧">Emitting the Initiator</a></li> 
<li><a href="#1-4" title="Photoshopの一覧">Embedding on the liposome</a></li></ul></li>
<br>
<li>The Motor
<ul><li><a href="#2-1" title="(X)HTMLの一覧">Folding the Motor-Monomer</a></li> 
<li><a href="#2-2" title="Web関連情報の一覧">Forming the Motor-Polymer</a></li></ul></li>
</ul>
<!--/ .category--></ul>
<!--/ #subContents--></div>
</head>
</head>


Line 114: Line 136:
<br>
<br>


<TABLE BORDER="0" WIDTH="810px" CELLSPACING="0" CELLPADDING="30">
<h1 class="title"><a name="background">&nbsp;Highlights</a></h1>
<TR>  
<br>
<TD align="center" BGCOLOR="#A9A9F5">  
 
<span style="font-size:40px; color:#0A0A2A;">The Receptor</span>
<div class="imagebox">
</TD>  
  <p class="image"><img src="http://openwetware.org/images/9/96/Koala_005.jpg" width="300" height="213"></p>
</TR>  
  <p class="caption">The agarose gel electrophoresis.</p>
</TABLE>  
</div>
<br clear="right">
 
<div class="smallbox">
  <p class="smallbox"><img src="http://openwetware.org/images/9/96/Koala_005.jpg" width=200px height="120px"></p>
  <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>
</div>
<div class="smallbox">
  <p class="simage"><img src="http://openwetware.org/images/9/96/Koala_005.jpg" width=200px height="120px"></p>
  <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>
</div>
<br clear="right">
 
<div class="centerbox">
  <p class="image"><img src="http://openwetware.org/images/9/96/Koala_005.jpg"></p>
  <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>
</div>
<div class="centerbox">
  <p class="image"><img src="http://openwetware.org/images/9/96/Koala_005.jpg"></p>
  <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>
</div>
<br clear="left">
 
<br>
<br>
<h1 class="title"><a name="background">&nbsp;Direction</a></h1>
<p class="paragraph">We planned to do experiments in several steps. First, we design the Receptor. Then, the following experiments are conducted in parallel.
</p>
<p class="paragraph">&#8226; Form the Receptor dimer by using thrombin.
<br>&#8226; Emit the Polymerization Initiator by demerization.
<br>&#8226; Embedding of Receptor on the liposome.</p>
<br>
<br>
<center><table><tr>
 
<td style="padding:0px 10px"><b>Forming the Receptor dimer</b></td><td style="padding:0px 80px"><b>Emiting the Initiator</b></td><td style="padding:0px 10px"><b>Embedding on the liposome</b></td>
<div class="CollapsibleBox" id="CBoxCover1">
</tr></table>
  <div class="CollapsibleBoxTitle">
<table><tr>
      <p class="cBoxButtons">
<td style="padding:0px 5px"><img src="http://openwetware.org/images/6/66/Receptorstep1.png" width="250px"></td>
        <a href="#" onclick="HideCBox('CBoxBody1');  return false;" title="折りたたみ／復元"><font color="white" font size="2">[show/hide]</font></a>
<td style="padding:0px 5px"><img src="http://openwetware.org/images/d/db/Receptorstep2.png" width="250px"></td>
      </p>
<td style="padding:0px 5px"><img src="http://openwetware.org/images/2/2e/Receptorstep3.png" width="250px"></td>
      <p class="cBoxTitle">&nbsp; Contents：</p>
</tr></table></center>
  </div>
<a name="1-1"></a>
  <div class="CollapsibleBoxBody" id="CBoxBody1">
      <ul style="list-style:none;">
        <li> <a href=#1> 1. The Sensing System: The Receptor </a>
        <ul style="list-style:none;">
            <li> <a href="#1-1">1-1. Preparing the components </a></li>
            <ul style="list-style:none;">
              <li> <a href="#1-1-1">1-1(a). Folding the Wall </a></li>
              <li> <a href="#1-1-2">1-1(b). Producing MISTIC </a></li>
              <li> <a href="#1-1-3">1-1(c). Designing the Activator </a></li>
            </ul>
            </li>
            <li> <a href="#1-2">1-2. Embedding the Wall into the liposome </a></li>
            <li> <a href="#1-3">1-3. Linking the Activator to the liposome </a></li>
            <li> <a href="#1-4">1-4. Combining the Wall and the Activator </a></li>
            <li> <a href="#1-5">1-5. Separating the Wall from the Activator </a></li>
        </ul>
        </li>
       
        <li> <a href="#2">2. The Moving System: The Motor </a>
        <ul style="list-style:none">
            <li> <a href="#2-1">2-1. Producing the Motor-Monomer </a>
            <ul style="list-style:none">
              <li> <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a></li>
              <li> <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a></li>
              <li> <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a></li>
            </ul>
            </li>
            <li> <a href="#2-2">2-2. Deactivating and reactivating the binding capacity of streptavidin </a></li>
            <li> <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a></li>
        </ul>
        </li>
      </ul>
  </div>
</div>
 
<a name="2"></a>
<br>
<br>
<p class="paragraph">Finally, after success in the experiments, we combine them to attain the dimerization and the Initiator emission on the liposome.</li>
<h1 class="title"><a name="background">&nbsp;The Receptor</a></h1>
 
<h1 class="sub">1-1. Preparing the components </h1>
<p class="paragraph">
Three componets were prepared to develop the Receptor: The Wall of DNA origami, MISTIC for embedding into the liposome and the Activator. Please check our <a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design">Design page</a> to check each component's role.</p>
 
<h1 class="big">1-1(a). Folding the Wall</h1>
 
<div class="smallbox">
<p class="smallimage"><img src="Time"></p>
<p class="caption">Fig.1-1(a)-1. Gel analysis of the Walls annealed in different time.</p>
</div>
<div class="smallbox">
<p class="smallimage"><img src="temperature"></p>
<p class="caption">Fig.1-1(a)-2. Gel analysis of the Walls annealed in different temperature.</p>
</div>
 
<p class="paragraph">In this experiment, the assembly condition of the Wall structure was optimized and results were analyzed by agarose gel electrophoresis.
The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.</p>
 
<br clear="right">
 
<div class="smallbox">
<p class="smallimage"><img src="NaCl"></p>
<p class="caption">Fig.1-1(a)-3. Gel analysis of the Walls annealed in different concentration of NaCl.</p>
</div>
<div class="smallbox">
<p class="smallimage"><img src="MgCls"></p>
<p class="caption">Fig.1-1(a)-4. Gel analysis of the Walls annealed in different concentration of MgCl2.</p>
</div>
 
<p class="paragraph">The optimum results were following:
<ul style="margin:5px 35px;">
<li>Optimum concentration of MgCl<sub>2</sub> is 15 mM.</li>
 
<li>Optimum temperature of annealing is 45.3 &deg;C.</li>
 
<li>Optimum time of annealing is 5 hours.</li>
 
<li>Optimum concentration of NaCl is 2.5 mM.</li>
</ul>
</p>
</p>
<br clear="right">
<br>
<br>
<h1 class="title"><a name="background">&nbsp;Experiments</a></h1>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(a)-5. TEM image of the Wall.</p>
</div>
 
<p class="paragraph">
The folding is corroborated by the TEM image (Fig.1-1-1.).
</p>
 
<br clear="right">
 
<h1 class="big">1-1(b). Producing MISTIC</h1>
<p class="paragraph">We introduced the variant to the gene of MISTIC, a protein which penetrates the membrane for 4 times, by using Quick Change method. The purpose of this experiment is to produce mono-cysteine MISTIC. By this operation, we would be able to label arbitrary position of MISTIC when we want to label cysteine. We introduced variation to valine which was at 29th from the initiation codon.</p>
<p class="paragraph">The wild type of the configuration of MISTIC was as follows.</p>
 
MFCTFFEKHHR<u>KWDILLEKST</u>GVMEAMK<font color="red">V</font>TS<u>EEKEQLSTAIDRMNEGLDAFIQLY</u>NESEIDEPLIQ
<u>LDDDTAELMKQARDMY</u>GQEK<u>LNEKLNTIIKQILSI</u>SVSEEGEKE
</p>
<p class="paragraph">
Underlined parts are considered to be the α-helix structures which penetrate membranes.
A red letter show the part which we introduced variation in this experiment.
</p>
<p class="paragraph">
We confirmed the introduction of variant by sequencing.
</p>
<p class="paragraph">
<b>(i) Preparation of templateDNA of MISTIC for PUREfrex</b>
</p>
<p class="paragraph">
In order to express MISTIC using protein synthesis using recombinant elements (PURE) frex, we introduced T7 promoter, SD sequence and T7 terminator to the variant DNA which we made. This introduction was conducted by PCR. PURE frex is a reconstructed cell-free system for transcription and translation reaction. At the same time, we substituted valine for cysteine3 and completed to make mono-cysteine configuration. We introduced Histag to the C-terminus and replaced Phe2 by amber, one of a termination codon. The latter introduction was done to introduce biotin to the N-terminus using unnatural amino acids.
</p>
<p class="paragraph">
Considering the efficiency of inserting unnatural amino acids would be influenced by the position of insertion, we made seven types of DNA and used the optimum replacement type of Phe2 to amber. Final configuration of MISTIC is as follows.</p>
<p>M+<font color="red">V</font>TFFEKHHRKWDILLEKSTGVMEAMK<font colos="red">C</font>TSEEKEQLSTAIDRMNEGLDAFIQLYNESEIDEPLIQLDDD
TAELMKQARDMYGQEKLNEKLNTIIKQILSISVSEEGEKE<font color="red">HHHHHH</font></p>
<p class="paragraph">+ is an abbreviation for unnatural amino acid.</p>
 
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(b)-1. Agarose gel electrophoresis of PCR products.</p>
</div>
<p class="paragraph">The completion of DNA for PURE frex was confirmed by gel electrophoresis. If the existence of the DNA which the length was the same as the one which we wanted was recognized, we resulted that the DNA was completed.</p>


<p class="paragraph">
As a single band was observed at the position we aimed (455bp), we resulted that DNA for PURE frex was completed.
</p>
<br clear="right">


<p class="paragraph">
<b>(ii) Expression and purification of MISTIC protein</b>
</p>


<h1 class="big">&nbsp;Folding the Receptor</h1>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(b)-2. SDS-PAGE of PURE products.</p>
</div>


<div class="left"><img src="http://openwetware.org/images/2/22/OCKtem.png"><img src="http://openwetware.org/images/1/14/Yui5.png" height="300px"></div>
<p class="paragraph">
<br>
We added MISTIC gene to the solution of PURE frex, incubated at 37&dig;C for 2 hours and formed MISTIC protein. The expression in PURE frex was confirmed by SDS-PAGE. In order to distinguish between the factor of PURE flex and MISTIC, we added 35S-Met to PURE flex and labelled MISTIC with a radioisotope and detected it by Photostimulated luminescence (PSL).
</p>
 
<p class="paragraph">
As we could observe the band at the aimed position (13kDa), we considered that MISTIC was formed in PURE flex.
</p>
<br clear="right">
 
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(b)-3. SDS-PAGE of PURE products and Purification Products.</p>
</div>
 
<p class="paragraph">We then purified a product of PURE flex using Histag column. The confirmation of the purification was done by SDS-PAGE.
</p>
 
<p class="paragraph">
As we could see MISTIC was included to the division of elusion and impurities were few, we resulted that the purification was succeeded.
</p>
<br clear="right">
 
<p class="paragraph">
<b>(iii) Confirmation of the introduction of unnatural amino acids</b>
</p>
 
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(b)-4. SDS-PAGE of PURE products</p>
</div>
 
<p class="paragraph">
By confirming that MISTIC forms when we used unnatural amino acids and cannot form without them, we would assay that that unnatural amino acids are included in MISTIC. We detected MISTIC labelled by a radioisotope by PSL (same method stated in <b>(iii)</b>). </p>
 
<p class="paragraph">
As we could see that MISTIC is not formed when unnatural amino acids are not added, we could determine that tRNA of amino acids which are inserted to the codon of amber competitively with RF1 does not exist. From this fact, we could confirm that unnatural amino acids are included in MISTIC including amber.
</p>
<br clear="right">
 
<p class="paragraph">
<b>(iv) Confirmation of the ability of access to biotin under the non-denatured condition</b>
</p>
 
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(b)-5. SDS-PAGE of PURE products</p>
</div>
 
<p class="paragraph">
By certifying whether or not MISTIC expressed in PURE frex are removed by the magnetic beads which streptavidin (SA) is connected, we confirmed that SA can access to niotin under the non-denatured condition. To exclude the influence of other PURE factors, we labelled it by 35S-Met and assayed by PSL.
</p>
 
<p class="paragraph">
We loaded same amount of the product of PURE to all lanes. As only the amount of MISTIC with biotin is decreased, biotin connected to MISTIC is able to be accessed and MISTIC was removed from solution through that connection.
</p>
<br clear="right">
 
<p class="paragraph">
<b>(v) Confirmation of modification of MISTIC to cysteine</b>
 
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(b)-6. SDS-PAGE of modified mistic (Cy3/SYPRO ORENGE)</p>
</div>
 
<p class="paragraph">
MISTIC was labelled by Cy3 maleimide (Cy3-MA) and confirmed whether cysteine residue of MISTIC is able to be modified or not. 0.45µL of 11mM Cy3-MA and 50µL of 3.26µM MISTIC were mixed and incubated for 30 minutes. Products were perdormed to electrophoresis using SDS-PAGE ageter purification and fluorescence was observed. After that, protein was dyed by SYPRO ORANGE and fluorescence was observed again.
</p>
 
<p class="paragraph">
As we could see the fluorescence of Cy3 at the position of the band of MISTIC, the modification seemed to be successful.
Comparing the intensity of fluorescence with other lanes which we put the same amount of Cy3-MA, the modification rate is presumed to be --%.
</p>
 
 
<h1 class="big">1-1(c). Designing the Activator</h1>
<p class="paragraph">
Two restriction enzymes, Hind&#8546; and Lambda Exonuclease, were compared to choose which is appropriate for the Activator. Two aspects were evaluated in this experiment: modification with oligonucleases and enzyme activity.</p>


<p class="ex">Folding of the Receptor was corroborated by TEM and 1% agarose gel electrophoresis analysis.
<p class="paragraph"><b>(i) Evaluating modification with oligonucleatides</b></p>
<br>The folded structure of the Receptor was clearly appeared in the TEM image.</p>


<a name="1-2">&nbsp;</a>
<div class="imagebox">
<br><br><br><br>
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(c)-1. BS(PEG)<sub>9</sub> for oligo-modification.</p>
</div>


<p class="paragraph">Hind&8546; and Lambda Exonuclease are modified with oligonucleotides to join the Activator and the Anchor. BS(PEG)<sub>9</sub> was used for the modification.</p>


<h1 class="big">&nbsp;Forming the Receptor dimer</h1>
<br clear="right">
<p class="ex">Before the experiment of forming the Receptor dimer with thrombin, we confirmed that thrombin-binding aptamers bind to thrombin. The molar ratio of the aptamers to thrombins was also optimized.</p>


<p class="ex">Binding of the aptamers with thrombin was analyzed with Native-PAGE.</p>
<p class="paragraph">On Hind&#8546;, the modification was analyzed by Native-PAGE. Oligonucleotides are labelled with Cy3 and Hind&#8546; is labelled with SYBR GREEN to confirm the modification.</p>


<p class="ex">The result showed that the aptamers should be added at least 10 times as many as alpha-thrombins for binding.</p>
<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(c)-2. Cy3 fluorescence image of oligo-modified Hind&#8546; by Native-PAGE.</p>
</div>


<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(c)-3. SYPRO-ORANGE fluorescence image of oligo-modified Hind&#8546; by Native-PAGE.</p>
</div>


<a name="1-3">&nbsp;</a>
<br clear="left">
<br><br><br><br>


<p class="paragraph">In the both figures, a band was observed in the same position in lane ○○. The modification of Hind3 with Cy3-oligonucleotides was therefore confirmed.</p>


<p class="paragraph">On Lamda Exonuclease, the modification was confirmed by Native-PAGE in the same way as Hind&#8546;.</p>


<h1 class="big">&nbsp;Emitting the Initiator</h1>
<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(c)-4. Cy3 fluorescence image of oligo-modified Lamda Exonuclease by Native-PAGE.</p>
</div>


<div class="right"><img src="http://openwetware.org/images/6/6b/Emittion.png"></div>
<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-1(c)-5. SYPRO-ORANGE fluorescence image of oligo-modified Lamda Exonuclease by Native-PAGE.</p>
</div>


<br><br><br><br>
<br clear="left">
<a name="1-4">&nbsp;</a>
<br><br><br><br>


<p class="paragraph">The result shows Lamda Exonuclease was modified with oligonucleotide successfully. </p>


<p class="paragraph"><b>(ii) Evaluating enzyme activity</b></p>
<div class="smallbox">
  <p class="smallimage"><img src=""></p>
  <p class="caption">Fig.1-1(c)-7. DNA cleavage reaction by Lamda Exonuclease.</p>
</div>
<div class="smallbox">
  <p class="smallimage"><img src=""></p>
  <p class="caption">Fig.1-1(c)-6. DNA cleavage reaction by Hind&#8546;.</p>
</div>
<p class="paragraph">
Hind&#8546; is an restriction endonuclease that recognizes base sequence 5’-AAGCTT-3’ and cleaves it, while Lambda Exonuclease degrades one strand from 5'-phosphoryl termini of dsDNA.</p>


<h1 class="big">&nbsp;Embedding on the liposome</h1>
<br clear="right">
<p class="paragraph">
In this experiment, enzyme activity of Hind&#8546; and Lambda Exonuclease was evaluated in various conditions. The purpose is to confirm that Hind&#8546; and Lambda Exonuclease have activity in condition for DNA origami. The activity was analyzed by Native-PAGE.</p>


<div class="right">
<div class="smallbox">
<img src="http://openwetware.org/images/b/bb/Yuichole.png" height="300px"></div>
  <p class="smallimage"><img src=""></p>
  <p class="caption">Fig.1-1(c)-9. Native-PAGE shows Lamda activity depending on PH.</p>
</div>


<br>
<div class="smallbox">
  <p class="smallimage"><img src=""></p>
  <p class="caption">Fig.1-1(c)-8. Native-PAGE shows Hind&#8546; activity depending on PH.</p>
</div>


<p class="ex">In order to certify the penetration of the Receptors to liposomes, a flotation assay was conducted.</p>
<p class="paragraph">
<p class="ex">In preparation for a floation assay, cholesterol oligomers were hybridized to the Receptors for penetration. The hybridization was confirmed by 1% agarose gel electrophoresis analysis.</p>
First, enzyme activity depending on PH was analyzed.</p>


<br><br><br><br><br><br><br><br>
<p class="paragraph">
The result showed that Lambda Exonuclease can act in pH7.5-8.5. In pH9.0 and pH9.4, dsDNA seems to be unstable. So, pH7.5-8.5 is appropriate for reaction of Lambda Exonuclease and DNA.
</p>


<div class="right"><img src="http://openwetware.org/images/d/d3/Flotation.png" height="250px"></div>
<br clear="right">


<br>
<div class="smallbox">
  <p class="smallimage"><img src=""></p>
  <p class="caption">Fig.1-1(c)-11. Native-PAGE shows Lamda activity depending on concentration of MgCl<sub>2</sub>.</p>
</div>


<p class="ex">In flotation assay, we divided mixture with the Receptors and the liposomes to a few fractions, each of them was analyzed by fluorescence spectrophotometer and 1% agarose gel electrophoresis. This assay showed in which fraction of the mixture includes the liposomes embedded by the Receptors.</p>
<div class="smallbox">
  <p class="smallimage"><img src=""></p>
  <p class="caption">Fig.1-1(c)-10. Native-PAGE shows Hind&#8546; activity depending on concentration of MgCl<sub>2</sub>.</p>
</div>


<br><br><br><br><br>
<p class="paragraph">
Then enzyme activity depending on concentration of MgCl<sub>2</sub> was analyzed.</p>
<p class="paragraph">
The result showed that Lambda Exonuclease can act in mM MgCl<b>2</b>.</p>


<div class="right"><img src="http://openwetware.org/images/8/8d/Keikou.JPG" width="300px"></div>
<br clear="right">


<br>
<h1 class="sub">1-2. Embedding the Wall into the liposome</h1>
<p class="paragraph">
Embedding the Wall into the liposome needs two steps: modifying the Wall with cholesterol and putting them into the liposome. The experiments were done individually.
</p>
<p class="paragraph">
(i) Hybridization of cholesterol oligomer with the Walls
</p>


<p class="ex"><b>The fluorescence intensity of NileRed in each fraction (NileRed dyes the membrane of liposomes.)</b></p>
<p class="paragraph">
<p class="ex">The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3.</p>
In this experiment, we examined the best concentration and the number of staples which the cholesterol oligomers can connect. Hybridization of cholesterol oligomer with the Walls is needed to penetrate the Walls into the membrane of liposomes. The result was assayed by 1% agarose gel electrophoresis.
</p>


<br><br><br><br><br><br><br>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-2-1. Confocal microscope image of GUV including Walls.</p>
</div>


<div class="left"><img src="http://openwetware.org/images/0/02/Yui3.png"><img src="http://openwetware.org/images/7/7e/Yui4.png"></div>
<p class="paragraph">
If the cholesterol oligomer was connected to the Walls the band would be smear.
In sample 2_1~4, 3_1, 3_2, 5_1, 5_2, 6_1.  
Considering that sample 2 and 3 do not have staple for hybridization, the Walls of sample 2 and 3 aggregated by the existence of cholesterol oligomer.
Sample 5_1, 5_2 and 6_1 are considered to be successful in hybridization. As the band of sample 5_2 and 6_1 are weaker than that of sample 5_1, we decided that the best condition was sample5_1.
</p>


<br><br><br><br><br><br><br>


<div class="right"><img src="http://openwetware.org/images/5/57/Yuigraph2.JPG" width="300px"></div>
<br clear="right">


<br><br><br>
<div class="imagebox">
<p class="ex"><b>The ratios of the Receptor in each fraction were analyzed by the density of band.</b></p>
  <p class="image"><img src=""></p>
<p class="ex">In sample1 (cholesterol + / liposome +), the ratio of the Receptor in fraction3 was a bit larger than that in fraction2. In contrast, in sample 2, 3 and 4, the ratio of the Receptor in fraction3 was smaller than that in fraction2. As liposomes were mostly seen in fraction3 in both sample 1 and 3, and the ratio of the Receptor in each fraction was different only in sample1, we concluded that this difference was caused by penetration of the Receptor to liposomes.</p>
  <p class="caption">Fig.1-2-1. Confocal microscope image of GUV including Walls.</p>
</div>


<p class="paragraph">
In this experiment, the Wall which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV) to penetrate the liposome from inside. Inclusion of Motor-Monomers and Walls was observed by confocal microscope.
</p>


<br clear="right">


The connection of Q-dot to the Wall was confirmed by agarose gel electrophoresis.


<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-2-2. Agarose gel electrophoresis showing the existence of Q-dots.</p>
</div>
<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.1-2-3. Agarose gel electrophoresis showing the existence of DNA.</p>
</div>
<br clear=left">


<br><br>
<p class="paragraph">
The first picture shows the existence of Q-dots and the second picture shows the existence of DNA. </p>
<p class="paragraph">
Comparing the seventh, eighth and ninth lane from the left in the first picture, we can see the difference in the position of the Q-dots. Comparing the eighth and eighth lane from the left in the two pictures, we can see the position of DNA, in this case, Walls, and the Q-dots in the same position. This data shows that the Q-dots connected to the Walls through the connection of biotin and streptavidin.
</p>




<h1 class="sub">1-3. Linking the Activator to the liposome</h1>


<h1 class="title"><a name="background">&nbsp;Next Steps</a></h1>
<h1 class="sub">1-4. Combining the Wall and the Activator</h1>
<p class="paragraph">In these experiments, we achieved to construct the Receptors and insert them into the liposomes. The very basics of others, formation of the Receptor dimers and emission of the Initiator, were also confirmed. Therefore, we are planning to approach the ones following for next steps. </p>
<p class="paragraph">&#8226; Confirm the more advanced mechanism of forming the Receptor dimers and emitting the Initiator.
<br>&#8226; Examine conditions for dimerization of the Receptor heterounits on the liposomes, may need changes in design of the Receptor.</p>


<h1 class="sub">1-5. Separating the Wall from the Activator</h1>


<a name="2">&nbsp;</a>
<a name="3"></a>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<h1 class="title"><a name="background">&nbsp;The Motor</a></h1>
<TABLE LEFTMARGIN="-10px" BORDER="0" WIDTH="810px" CELLSPACING="0" CELLPADDING="30">
 
<TR>
<h1 class="sub">2-1. Producing the Motor-Monomer </h1>
<TD align="center" BGCOLOR="#A9A9F5">
<span style="font-size:40px; color:#0A0A2A;">The Motor Monomer</span>
</TD>
</TR>
</TABLE>
<br>
<h1 class="title"><a name="background">&nbsp;Direction</a></h1>
<br>
<p class="paragraph">We planned to do experiments in several steps. First, we design the Motor-Monomer and assay the formation of a simple Motor-Polymer. Then, the following experiments are conducted in parallel.</p>
<p class="paragraph">&#8226; Control the initiation of the polymerization (forming ring structures and ring-opening polymerization).
<br>&#8226; Put the Motor-Monomers into the liposome.</p>


<br>
<p class="paragraph">
<center><table><tr>
Producing the Motor-Monomer is divided into three sections: Folding the Motor-Monomer body, synthesizing divalent streptavidin and equipping the Motor-Monomer body with divalent streptavidin.
<td style="padding:0px 80px"><b>Controlling the polymerization</b></td><td style="padding:0px 80px"><b>Putting into the liposome</b></td>
</p>
</tr></table>
<table><tr>
<td style="padding:0px 30px"><img src="http://openwetware.org/images/9/91/Monomerstep1.png" width="400px"></td>
<td style="padding:0px 30px"><img src="http://openwetware.org/images/2/28/Monomerstep2.png" width="300px"></td>
</tr></table></center>


<a name="2-1"></a>
<h1 class="big">2-1(a). Folding the Motor-Monomer body
<p class="paragraph">Finally, after success in the experiments, we combine them to attain the polymerization in the liposome.</p>
</h1>
<br><br>
<div class="smallbox">
<p class="smallimage"><img src="Time"></p>
<p class="caption">Fig.2-1(a)-1. Gel analysis of the Monomers annealed in different time.</p>
</div>
<div class="smallbox">
<p class="smallimage"><img src="temperature"></p>
<p class="caption">Fig.2-1(a)-2. Gel analysis of the Monomers annealed in different temperature.</p>
</div>


<p class="paragraph">In this experiment, the assembly condition of the Motor-Monomer structure was optimized and results were analyzed by agarose gel electrophoresis. The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.</p>


<h1 class="title"><a name="background">&nbsp;Experiments</a></h1>
<br clear="right">


<div class="smallbox">
<p class="smallimage"><img src="NaCl"></p>
<p class="caption">Fig.2-1(a)-3. Gel analysis of the Monomers annealed in different concentration of NaCl.</p>
</div>
<div class="smallbox">
<p class="smallimage"><img src="MgCls"></p>
<p class="caption">Fig.2-1(a)-4. Gel analysis of the Monomers annealed in different concentration of MgCl2.</p>
</div>


<p class="paragraph">The optimum results were following:
<ul style="margin:5px 35px;">
<li>Optimum concentration of MgCl<sub>2</sub> is 15 mM.</li>


<h1 class="big">&nbsp;Folding the Motor-Monomer</h1>
<li>Optimum temperature of annealing is 45.3 &deg;C.</li>


<div class="right"><img src="http://openwetware.org/images/b/b6/Monomerjouken.png"></div>
<li>Optimum time of annealing is 5 hours.</li>


<p class="ex">The assembly condition of the Motor-Monomer was optimezed with concentration of MgCl<sub>2</sub>, annealing temperature and time. The resulting structure was analyzed by agarose gel electrophoresis.</p>
<li>Optimum concentration of NaCl is 2.5 mM.</li>
</ul>
</p>
<br clear="right">


<br>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1(a)-5. TEM image for the Motor-Monomers.</p>
</div>


<div class="right"><img src="http://openwetware.org/images/b/bc/2-1-1.png" width="300px" height="300px"></div>
<p class="paragraph">
The folding is corroborated by the TEM image.
</p>
<br clear="right">


<br>
<p class="paragraph">
<p class="ex">Multimers (e.g. dimmers) were appeared at lower temperature, so optimum temperature of annealing was 55.0&#8451;.</p>


<br><br><br><br><br><br><br><br><br><br>


<h1 class="big">2-1(b). Synthesizing divalent streptavidin</h1>


<div class="right"><img src="http://openwetware.org/images/4/48/2-1-2.png" height="300px"><img src="http://openwetware.org/images/e/e2/2-1-3.png"></div>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1(b)-1. SDS-PAGE showing purified divalent SA.</p>
</div>
<p class="paragraph">
In this experiment, divalent streptavidin (SA) was synthesized for equipment to the Motor-Monomers.
</p>
<p class="paragraph">
TaKaRa Competent Cell BL21 was used for protein expression. The purification of divalent SA on the nickel-affinity column was analyzed by SDS-PAGE.
</p>  
<br clear="right">


<p class="ex">(Annealing temperature : 55.0&#8451;, M13 : staples = 1 : 2)
<div class="imagebox">
<br>The band of monomer at 16mM is bending and its migration distance is different from the one of MoN. It shows that the Monomers were not annealed correctly at 16 mM. There seemed to be little difference at other concentration, so optimum concentration of MgCl<sub>2</sub> was the range 10  to 14mM.</p>
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1(b)-2. SDS-PAGE showing the affinity of divalent SA with biotin-modified oligos.</p>
</div>
The affinity of divalent SA with biotin-modified oligonucleotides was then analyzed by SDS-PAGE.
<br clear="right">


<br><br><br>
<h1 class="big">2-1(c). Equipping the Motor-Monomer with SA</h1>
<p class="paragraph">
Because linking the Motor-Monomer and divalent SA uses modification of divalent SA by NHS ester reaction and click reaction, SA was linked with cy5-modified oligonucleotide via click reaction and NHS ester reaction, then the ability of modified SA to bind biotin-nucleotide was confirmed in this experiment.</p>


<div class="right"><img src="http://openwetware.org/images/f/f4/2-1-4.png"><img src="http://openwetware.org/images/f/fa/2-1-5.png"></div>


<p class="ex">(Annealing temperature : 55.0&#8451;, MgCl<sub>2</sub> : 10mM)
<div class="centerbox">
<br>At the ratio of 6 and 8 a band of a monomer is weak and migrating distance was shorter than the one of monomer. As the graph showed, the ratio of dimer to monomer was low in lane 1 and 2, so optimum ratio of staples to scaffold was 2 or 4.</p>
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1-3. .</p>
</div>
<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1-4. .</p>
</div>
<br clear="left">


<br><br>
<p class="paragraph">
Figure 2-1-3 shows that SA was linked with cy5-modified oligonucleotide via click reaction. In Fig.2-1-4, because the lane of only Cy5 and NH2-modified oligonucleotide is not stained, the stained bands is from biotin-oligonucleotide. This picture shows modified SA can bind biotin. It may show the ability to bind biotin decreases as the amount of modification of SA increases, but the experiment of modification SA with Cy5-NHS deny. The reason of that may be derived from steric hindrance of Cy5-oligonucleotide which binds to SA.</p>
<p class="paragraph">
These figures confirm the ability of modified SA to bind biotin-nucleotide.</p>


<a name="2-2"></a>
<p class="ex">Folding of the Motor-Monomer was corroborated by PAGE analysis. The folding, however, was not verified by TEM. We considered it was because of hollow structures of the Motor-Monomers.</p>


<br>
<br>


<h1 class="big">&nbsp;Forming the Motor-Polymer</h1>
<h1 class="sub">2-2. Deactivating and activating the binding capacity of streptavidin </h1>


<div class="left"><img src="http://openwetware.org/images/9/90/Poly1.png" height="300px"><img src="http://openwetware.org/images/b/b0/Poly4.png" weight="300px" height="300px"></div>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-2-1. Mechanism of this experiment.</p>
</div>


<div class="left"><img src="http://openwetware.org/images/a/a5/Poly2.png"><img src="http://openwetware.org/images/3/3c/Poly5.png"></div>
<p class="paragraph">
In this experiment, deactivation and reactivation of streptavidin (SA) were confirmed by Native PAGE.
</p>
<p class="paragraph">First, two complementary oligonucleotides having biotin or desthiobiotin were annealed. The oligos having biotin is named Linker and the oligos having desthiobiotin is named Blocker. The resulting dsDNA was then mixed with divalent SA for deactivation. After removing excess SA with biotin-beads, ds-DNA bound to divalent SA was cut by Hind&#8546; for reactivation. Another oligonucleotides having biotin, named Chaser, were then added to replace Blocker.</p>


<br><br><br>
<p class="paragraph">Blocker was labeled with Cy3 and Chaser was labeled with Cy5 to confirm the replacement by Native-PAGE.</p>
<p class="ex">In this experiment, the effect of arm number on polymerization was examined. Two samples, one has three arms extruded from the body and the other has one, was compared. The Monomers comprise two similar structures, the Motor-Monomer A and the Motor-Monomer B. The polymerization was confirmed by mixing the Monomer A and the Monomer B.</p>
<br clear="right">


<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-2-2. Cy3 fluorescence image of Native-PAGE.</p>
</div>
<div class="centerbox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-2-3. Cy5 fluorescence image of Native-PAGE .</p>
</div>
<br clear="left">


<p class="ex">The ratio of dimers of three arms monomers roughly increased with time, while the ratio did not change significantly for one arm monomers. It confirmed the polymerization of the Monomers with three arms.</p>
<p class="paragraph">
In fig.2-2-3, multiple bands of SA-dsDNA complexes appears in lane 3 which is caused by non-ideal complexes. It is considered that biotin-beads successfully distinguished the ideal SA-dsDNA complex because the upper band in lane 3 mostly disappeared in lane 4.</p>
<p class="paragraph">
In fig.2-2-4, a band at the same position of the ideal SA-dsDNA complex is only shown in lane 7. We therefore confirmed the replacement of Blocker with Chaser after cutting dsDNA by Hind&#8546; and that the replacement did not happen without Hind&#8546;.</p>


<div class="left"><img src="http://openwetware.org/images/f/f1/Poly7.png" height="300px"><img src="http://openwetware.org/images/9/94/Poly9.png" height="300px"></div>
<h1 class="sub">2-3. Putting the Motor-Monomers into the liposome </h1>
<div class="left"><img src="http://openwetware.org/images/4/4f/Poly8.png"><img src="http://openwetware.org/images/e/e7/Poly10.png"></div>


<br><br><br>
<p class="paragraph">
<p class="ex">The Monomer concentration was optimized.</p>
In this experiment, Motor-Monomers which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV). Inclusion of Motor-Monomers was observed by confocal microscope.
<p class="ex">The ratio of dimers was high at high monomer concentration of three arms monomers, while for one arm monomers, the ratio was not different from that of monomer only condition. It meant Polymerization of three arms monomers proceeds efficiently at high concentration of monomers in solution.</p>
</p>
<p class="paragraph">
GUVs were dyed by Nile Red. Yellow green dots indicate the fluorescence of Nile Red and red dots indicate the fluorescence of Q-dots combined with Motor-Monomers.
</p>


<center>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-3-1. Confocal microscope image of GUV including Motor-Monomers.</p>
</div>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1-2. Confocal microscope image of GUV not including Motor-Monomers.</p>
</div>
</center>
<br clear="right">


<p class="paragraph">
Comparing the two pictures, we can see the red dots inside the GUV only in the picture showing the GUVs containing Motor-Monomers in the inner solution. From this fact, we could confirm the inclusion of Motor-Monomers into the GUV. As the excitation and fluorescence wave length of Nile Red and Q-dots were close, the membrane of GUV was shown in red even in the picture of GUV not including the Q-dots.
</p>
<br clear="right">


<p class="paragraph">
The connection of Q-dot to the Motor-Monomer was then confirmed by the agarose gel electrophoresis.
</p>


<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1-3. Agarose gel electrophoresis showing existence of Q-dots.</p>
</div>
<div class="imagebox">
  <p class="image"><img src=""></p>
  <p class="caption">Fig.2-1-3. Agarose gel electrophoresis showing existence of DNA.</p>
</div>
<br clear="right">


<p class="paragraph">
The first picture shows the existence of Q-dots and the second picture shows the existence of DNA. Comparing the fourth, fifth and sixth lane from the left in the first picture, we can see the difference in the position of the Q-dots. Comparing the fifth and sixth lane from the left in the two pictures, we can see the position of DNA, in this case, Motor-Monomers, and the Q-dots in the same position. This data shows that the Q-dots connected to the Motor-Monomers through the connection of biotin and streptavidin.
</p>




<br>
<br>
<br>
<h1 class="title"><a name="background">&nbsp;Next Steps</a></h1>
<br>


<br>
 
<br>
<h1 class="title">Reference</h1>
</body>
</body>


Latest revision as of 02:57, 25 October 2014

<html> <head> <div id="#top"></div>

<script type="text/javascript" src="http://code.jquery.com/jquery-1.9.1.min.js"></script>

<script type="text/javascript"> new Image(120,80) = "http://openwetware.org/images/7/7a/Logo2Kashiwa.png";

new image(120,80) = "http://openwetware.org/images/1/1d/Logo2.5.png";

new image(71,143) = "http://openwetware.org/images/2/2a/Top1.gif";

new image(71,143) = "http://openwetware.org/images/f/f0/Top2.gif";

new image() = "http://openwetware.org/images/3/38/BackgroundKashiwa.png";

new image() = "http://openwetware.org/images/9/9c/BackgroundKashiwa2.png"; </script>


<script type="text/javascript"> 

  $(function(){
     $("#menu li").hover(function(){
        $("ul",this).show();
     },
     function(){
        $("ul",this).hide();
     });
  });

</script>


<script type="text/javascript"> <!-- if (document.images) {

// 設定開始（使用する画像を設定してください）

// 通常の画像 var img0 = new Image(120,80); img0.src = "http://openwetware.org/images/e/ec/LogoKashiwa.png";

// ポイント時の画像1 var img1 = new Image(120,80); img1.src = "http://openwetware.org/images/2/26/Logo3Kashiwa.png";


// 設定終了

} // ポイント時の処理 function On(name) { if (document.images) { document.images['def'].src = eval(name + '.src'); } } // 放した時の処理 function Off() { if (document.images) { document.images['def'].src = img0.src; } } // --> </script>

<META http-equiv="Content-Style-Type" content="text/css">

<style type="text/css">


.page-back { position: fixed; bottom: 25px; right: 25px;}

p.paragraph { 

 font-size :100%;
 line-height: 135%;
 margin: 10px 25px;

}

p.reference { 

 font-size :90%;
 line-height: 135%;
 margin: 10px 20px;

}

p.indent-paragraph { 

 font-size :110%;
 line-height:1.5;
 margin:0 30px;
 text-indent: 1em;
 }

h1.title a{ 

 font-size :100%;
 display: block;
 text-decoration: none;
 color: #000000;

　font-weight:bolder; 

 border-left: solid 5px #e00000;
 }

h2.reference a{ 

 font-size :90%;
 display: block;
 text-decoration: none;
 border-left: solid 5px;
 }

body { 

 font-size: 12px;
 font-family: Frutiger, Helvetica, Arial;
 background-color: #B5D3FF;
 overflow-y:scroll;
 overflow-x:hidden;

} article { 

 background-color: #ffffff

} .container { 

  background-color: #ffffff;
  margin-top:0px

}

.OWWNBcpCurrentDateFilled {display: none;}

}

  1. column-content

{ 

  width: 0px;
  margin: 0;
  padding: 0;
  float:left;

} .firstHeading { 

  display:none;
  width:0px;

}

  1. globalWrapper

{ 

  width:900px;
  background-color: #ffffff;
  margin-left: auto;
  margin-right: auto;

}

  1. column-one

{ 

  display:none;
  width:0px;
  background-color: #ffffff;

}

  1. content

{ 

  margin: 0px;
  align: center;
  padding: 0px 12px;
  width:876px;
  border: 0;

}

  1. bodyContent

{ 

  width: 800px;
  padding: 0px 38px;
  align: center;
  background-color: #ffffff;
  position:relative;

}

  1. column-content

{ 

  width: 900px;
  background-color: #ffffff;

}


  1. footer

{ 

  position: center;
  width: 900px;

}

div.menubar { 

  position: fixed;
  background-color: none; /* バーの背景色 */
  opacity: 0.95;
  border-top: 0px double white; /* バーの上端線 */
  border-bottom: 0px double white; /* バーの下端線 */
  min-width: 900px; /* メインメニュー全部が収まる最低横幅 */
  z-index:3

}

div.menubar ul#menu { 

  margin: -33px 0px 0px -50px; /* メニューバー外側の余白 */
  padding: 0px; /* メニューバー内側の余白 */
  height: 80px; /* メニューバーの高さ */
  list-style-type: none;

}

div.menubar ul#menu li { 

  min-width: 110px; /* メニュー項目の横幅 */
  height: 80px; /* メニュー項目の高さ(「メニューバーの高さ」と一致させる) */ 
  float: left;
  list-style-type: none;
  list-style-image: none;
  position: relative;

}

div.menubar ul#menu a { 

  text-decoration: none; /* メニュー項目の装飾(下線を消す) */
  display: block; 
  background-color: #1F003E;/* メニュー項目の背景色 */
  color: white; /* メニュー項目の文字色 */
  line-height: 80px; /* メニュー項目のリンクの高さ(「メニュー項目の高さ」と一致させる) */
  text-align: center; /* メインメニューの文字列の配置(中央寄せ) */　
  width: 100%;
  height: 100%;

}

div.menubar ul#menu a:hover { 

  background-image: url("http://openwetware.org/images/3/38/BackgroundKashiwa.png"); /* メニュー項目にマウスが載ったときの背景色 */
  color: #8B008B; /* メニュー項目にマウスが載ったときの文字色 */

}

div.menubar ul#menu a:active { 

  background-image: url("http://openwetware.org/images/3/33/Background2.png"); /* メニュー項目にマウスが載ったときの背景色 */
  color: #8B008B; /* メニュー項目をクリックした時の文字色 */

}

/* メニューバー直後のClearfix */ div.menubar ul#menu { 

  zoom:1;

} div.menubar ul#menu:after { 

  height: 0; visibility: hidden; content: "."; display: block; clear: both;

}

div.menubar ul#menu ul.sub { 

  background-color: #1F003E; /* サブメニュー全体の背景色 */
  border-width: 0px 0px 0px 0px; /* サブメニュー全体の枠線の太さ */
  border-style: solid; /* サブメニュー全体の枠線の線種 */
  border-color: #191970; /* サブメニュー全体の枠線の色 */
  margin: 0px;
  padding: 0px;
  display: none;
  position: absolute;

}

div.menubar ul#menu ul.sub li { 

  width: 110px;
  height: 50px; /* サブメニュー1項目の高さ */
  border-width: 0px 0px 0px 0px; /* サブメニュー1項目の枠線の太さ */
  border-style: solid; /* サブメニュー1項目の枠線の線種 */
  border-color: #191970; /* サブメニュー1項目の枠線の色 */
  z-index: 3

}

div.menubar ul#menu ul.sub li a { 

  line-height: 50px; /* サブメニュー1項目の行の高さ(「サブメニュー1項目の高さ」と合わせる) */
  text-align: center;  /* サブメニュー1項目の項目名の配置(中央寄せ) */
  text-indent: 0px;  /* サブメニュー1項目の項目名前方の余白 */

}

div.menubar ul#menu ul.sub li a:hover { 

  background-image: ('back');
  background-size: 100% auto;
  background-color: #FF00FF; /* サブメニュー項目にマウスが載ったときの背景色 */
  color:  #8B008B; /* サブメニュー項目にマウスが載ったときの文字色 */

}

div.menubar ul#menu ul.sub li a:active { 

  background-image: ('backclick') /* メニュー項目にマウスが載ったときの背景色 */
  backround-size: 100% auto;
  color: #8B008B; /* メニュー項目をクリックした時の文字色 */

}

</style>

<div class="menubar"> 

 <ul id="menu">
 <font face="Frutiger,Helvetica,Arial">
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa"><img src="http://openwetware.org/images/e/ec/LogoKashiwa.png" onmouseover="this.src='http://openwetware.org/images/7/7a/Logo2Kashiwa.png'" onclick="this.src='http://openwetware.org/images/1/1d/Logo2.5.png'" onmouseout="this.src='http://openwetware.org/images/e/ec/LogoKashiwa.png'" height="80px" width="120px" name="def"></a>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROJECT</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Background</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Motivation</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#3" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Project Goals</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EARLY TRIAL&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Design</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Approaches</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">DESIGN</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EXPERIMENT&nbsp;&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Highlights</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Receptor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Motor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li>
     </ul>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;&nbsp;DISCUSSION&nbsp;</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Achievements</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Future</span></a></li>
     </ul>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Protocols" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROTOCOL</span></a>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">TEAM</span></a>
     <ul class="sub">
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Members</span></a></li>
       <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Sponsors</span></a></li>
     </ul>
   </li>
 </font>
 </ul>

</div> <br> <br> <br> <div class="page-back"><a href="#top"> <img src="http://openwetware.org/images/2/2a/Top1.gif" onmouseover="this.src='http://openwetware.org/images/f/f0/Top2.gif'" onmouseout="this.src='http://openwetware.org/images/2/2a/Top1.gif'" height="150px"/></a></div> </head>


</html>

<html>

<head> <style>

div.imagebox { 

  border: 1.7px dashed #2E2EFE; /* 1.枠線 */
  background-color: #F0F8FF;  /* 2.背景色 */
  width: 310px; /* 横幅 */
  float: right; /* 右に配置 */
  margin: 0px 30px 0px 10px;

} p.image { 

  text-align: center;
  width: 300px;
  height: 213px;
  margin: 5px;

} p.caption { 

  margin: 5px 10px;
  font-size: 90%;  /* 5.文字サイズ */
  color: black; /* 6.文字色 */

}

div.centerbox { 

  border: 1.7px dashed #2E2EFE; /* 1.枠線 */
  background-color: #F0F8FF;  /* 2.背景色 */
  width: 310px; /* 横幅 */
  margin: 0px 40px;
  float: left;

}

div.smallbox { 

  border: 1.7px dashed #2E2EFE; /* 1.枠線 */
  background-color: #F0F8FF;  /* 2.背景色 */
  width: 210px; /* 横幅 */
  margin: 5px;
  float: right;

} p.smallimage { 

  text-align: center;
  width: 200px;
  height: 120px;
  margin: 5px;

}

h1.big{ 

 font-size :16px;
 text-decoration: none;
 color: #000000;
 margin: 10px 15px;
 font-weight: bolder;
 }

h1.sub { 

 font-size:20px;
 font-weight: bolder;
 margin: 10px 5px;

}

p.menu { 

 font-size :100%;
 line-height: 135%;
 margin: 0px 40px;

}

</style>

<script type="text/javascript"><!-- function HideCBox( boxid ) { 

  var target = document.getElementById(boxid);
  if( target.style.display != "none" ) {
     target.style.display = "none";
  }
  else {
     target.style.display = "";
  }

} // --></script>

<style type="text/css"><!-- div.CollapsibleBox { 

  /* ■ボックス全体■ */
  border: 0px solid #4169E1; /* 枠線:濃青 */
  font-size: 100%;             /* 文字サイズ:100% */
  width: 600px;               /* 横幅:600px */

} div.CollapsibleBox div.CollapsibleBoxTitle { 

  /* ■タイトル枠■ */
  background-color: #191970; /* 背景色:濃青 */
  color: white;               /* 文字色:白 */
  margin: 0px;
  padding: 0px;
  width: 600px;

} div.CollapsibleBox div.CollapsibleBoxTitle p.cBoxTitle { 

  /* ■タイトル文字列■ */
  font-size: 100% ;          /* 太字 */
  border: 1px solid #4B0082; /* 枠線:濃青 */
  margin: 0px;
  padding: 1px 0px;

} div.CollapsibleBox div.CollapsibleBoxTitle p.cBoxButtons { 

  /* ■ボタン群■ */
  margin: 0px;
  padding: 2px 10px;
  float: right;
  position: relative;

} div.CollapsibleBox div.CollapsibleBoxTitle p.cBoxButtons a img { 

  /* ■ボタンそのもの■ */
  border-width: 0px;    /* リンクの枠線を消す */

} div.CollapsibleBox div.CollapsibleBoxBody { 

  /* ■本文枠■ */
  margin: 0px;
  padding: 2px 0px 2px 0px;
  background-color: #ADD8E6; /* 背景色:淡青 */
  color: #191970; /* 文字色:暗青 */

} div.CollapsibleBox div.CollapsibleBoxBody p { 

  /* ■本文■ */
  margin: 0px;
  padding: 0.5em;   /* 内側の余白:0.5文字分 */

}

</style> </head>

<body> <a name="1"></a> <font face="Futura,Arial,Frutiger" font size="24px">EXPERIMENTS</font> <br> <br>

<h1 class="title"><a name="background">&nbsp;Highlights</a></h1> <br>

<div class="imagebox"> 

  <p class="image"><img src="http://openwetware.org/images/9/96/Koala_005.jpg" width="300" height="213"></p>
  <p class="caption">The agarose gel electrophoresis.</p>

</div> <br clear="right">

<div class="smallbox"> 

 <p class="smallbox"><img src="http://openwetware.org/images/9/96/Koala_005.jpg" width=200px height="120px"></p>
 <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>

</div> <div class="smallbox"> 

 <p class="simage"><img src="http://openwetware.org/images/9/96/Koala_005.jpg" width=200px height="120px"></p>
 <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>

</div> <br clear="right">

<div class="centerbox"> 

 <p class="image"><img src="http://openwetware.org/images/9/96/Koala_005.jpg"></p>
 <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>

</div> <div class="centerbox"> 

 <p class="image"><img src="http://openwetware.org/images/9/96/Koala_005.jpg"></p>
 <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>

</div> <br clear="left">

<br> <br>

<div class="CollapsibleBox" id="CBoxCover1"> 

  <div class="CollapsibleBoxTitle">
     <p class="cBoxButtons">
        <a href="#" onclick="HideCBox('CBoxBody1');  return false;" title="折りたたみ／復元"><font color="white" font size="2">[show/hide]</font></a>
     </p>
     <p class="cBoxTitle">&nbsp; Contents：</p>
  </div>
  <div class="CollapsibleBoxBody" id="CBoxBody1">
     <ul style="list-style:none;">
        <li> <a href=#1> 1. The Sensing System: The Receptor </a>
        <ul style="list-style:none;">
           <li> <a href="#1-1">1-1. Preparing the components </a></li>
           <ul style="list-style:none;">
              <li> <a href="#1-1-1">1-1(a). Folding the Wall </a></li>
              <li> <a href="#1-1-2">1-1(b). Producing MISTIC </a></li>
              <li> <a href="#1-1-3">1-1(c). Designing the Activator </a></li>
           </ul>
           </li>
           <li> <a href="#1-2">1-2. Embedding the Wall into the liposome </a></li>
           <li> <a href="#1-3">1-3. Linking the Activator to the liposome </a></li>
           <li> <a href="#1-4">1-4. Combining the Wall and the Activator </a></li>
           <li> <a href="#1-5">1-5. Separating the Wall from the Activator </a></li>
        </ul>
        </li>
        
        <li> <a href="#2">2. The Moving System: The Motor </a>
        <ul style="list-style:none">
           <li> <a href="#2-1">2-1. Producing the Motor-Monomer </a>
           <ul style="list-style:none">
             <li> <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a></li>
             <li> <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a></li>
             <li> <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a></li>
           </ul>
           </li>
           <li> <a href="#2-2">2-2. Deactivating and reactivating the binding capacity of streptavidin </a></li>
           <li> <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a></li>
        </ul>
        </li>
     </ul>
  </div>

</div>

<a name="2"></a> <br> <h1 class="title"><a name="background">&nbsp;The Receptor</a></h1>

<h1 class="sub">1-1. Preparing the components </h1> <p class="paragraph"> Three componets were prepared to develop the Receptor: The Wall of DNA origami, MISTIC for embedding into the liposome and the Activator. Please check our <a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design">Design page</a> to check each component's role.</p>

<h1 class="big">1-1(a). Folding the Wall</h1>

<div class="smallbox"> <p class="smallimage"><img src="Time"></p> <p class="caption">Fig.1-1(a)-1. Gel analysis of the Walls annealed in different time.</p> </div> <div class="smallbox"> <p class="smallimage"><img src="temperature"></p> <p class="caption">Fig.1-1(a)-2. Gel analysis of the Walls annealed in different temperature.</p> </div>

<p class="paragraph">In this experiment, the assembly condition of the Wall structure was optimized and results were analyzed by agarose gel electrophoresis. 

The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.</p>

<br clear="right">

<div class="smallbox"> <p class="smallimage"><img src="NaCl"></p> <p class="caption">Fig.1-1(a)-3. Gel analysis of the Walls annealed in different concentration of NaCl.</p> </div> <div class="smallbox"> <p class="smallimage"><img src="MgCls"></p> <p class="caption">Fig.1-1(a)-4. Gel analysis of the Walls annealed in different concentration of MgCl2.</p> </div>

<p class="paragraph">The optimum results were following: <ul style="margin:5px 35px;"> <li>Optimum concentration of MgCl<sub>2</sub> is 15 mM.</li>

<li>Optimum temperature of annealing is 45.3 &deg;C.</li>

<li>Optimum time of annealing is 5 hours.</li>

<li>Optimum concentration of NaCl is 2.5 mM.</li> </ul> </p>

<br clear="right">


<br> <div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(a)-5. TEM image of the Wall.</p>

</div>

<p class="paragraph"> The folding is corroborated by the TEM image (Fig.1-1-1.). </p>

<br clear="right">

<h1 class="big">1-1(b). Producing MISTIC</h1> <p class="paragraph">We introduced the variant to the gene of MISTIC, a protein which penetrates the membrane for 4 times, by using Quick Change method. The purpose of this experiment is to produce mono-cysteine MISTIC. By this operation, we would be able to label arbitrary position of MISTIC when we want to label cysteine. We introduced variation to valine which was at 29th from the initiation codon.</p> <p class="paragraph">The wild type of the configuration of MISTIC was as follows.</p>

MFCTFFEKHHR<u>KWDILLEKST</u>GVMEAMK<font color="red">V</font>TS<u>EEKEQLSTAIDRMNEGLDAFIQLY</u>NESEIDEPLIQ <u>LDDDTAELMKQARDMY</u>GQEK<u>LNEKLNTIIKQILSI</u>SVSEEGEKE </p> <p class="paragraph"> Underlined parts are considered to be the α-helix structures which penetrate membranes. A red letter show the part which we introduced variation in this experiment. </p> <p class="paragraph"> We confirmed the introduction of variant by sequencing. </p> <p class="paragraph"> <b>(i) Preparation of templateDNA of MISTIC for PUREfrex</b> </p> <p class="paragraph"> In order to express MISTIC using protein synthesis using recombinant elements (PURE) frex, we introduced T7 promoter, SD sequence and T7 terminator to the variant DNA which we made. This introduction was conducted by PCR. PURE frex is a reconstructed cell-free system for transcription and translation reaction. At the same time, we substituted valine for cysteine3 and completed to make mono-cysteine configuration. We introduced Histag to the C-terminus and replaced Phe2 by amber, one of a termination codon. The latter introduction was done to introduce biotin to the N-terminus using unnatural amino acids. </p> <p class="paragraph"> Considering the efficiency of inserting unnatural amino acids would be influenced by the position of insertion, we made seven types of DNA and used the optimum replacement type of Phe2 to amber. Final configuration of MISTIC is as follows.</p> <p>M+<font color="red">V</font>TFFEKHHRKWDILLEKSTGVMEAMK<font colos="red">C</font>TSEEKEQLSTAIDRMNEGLDAFIQLYNESEIDEPLIQLDDD TAELMKQARDMYGQEKLNEKLNTIIKQILSISVSEEGEKE<font color="red">HHHHHH</font></p> <p class="paragraph">+ is an abbreviation for unnatural amino acid.</p>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(b)-1. Agarose gel electrophoresis of PCR products.</p>

</div> <p class="paragraph">The completion of DNA for PURE frex was confirmed by gel electrophoresis. If the existence of the DNA which the length was the same as the one which we wanted was recognized, we resulted that the DNA was completed.</p>

<p class="paragraph"> As a single band was observed at the position we aimed (455bp), we resulted that DNA for PURE frex was completed. </p> <br clear="right">

<p class="paragraph"> <b>(ii) Expression and purification of MISTIC protein</b> </p>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(b)-2. SDS-PAGE of PURE products.</p>

</div>

<p class="paragraph"> We added MISTIC gene to the solution of PURE frex, incubated at 37&dig;C for 2 hours and formed MISTIC protein. The expression in PURE frex was confirmed by SDS-PAGE. In order to distinguish between the factor of PURE flex and MISTIC, we added 35S-Met to PURE flex and labelled MISTIC with a radioisotope and detected it by Photostimulated luminescence (PSL). </p>

<p class="paragraph"> As we could observe the band at the aimed position (13kDa), we considered that MISTIC was formed in PURE flex. </p> <br clear="right">

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(b)-3. SDS-PAGE of PURE products and Purification Products.</p>

</div>

<p class="paragraph">We then purified a product of PURE flex using Histag column. The confirmation of the purification was done by SDS-PAGE. </p>

<p class="paragraph"> As we could see MISTIC was included to the division of elusion and impurities were few, we resulted that the purification was succeeded. </p> <br clear="right">

<p class="paragraph"> <b>(iii) Confirmation of the introduction of unnatural amino acids</b> </p>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(b)-4. SDS-PAGE of PURE products</p>

</div>

<p class="paragraph"> By confirming that MISTIC forms when we used unnatural amino acids and cannot form without them, we would assay that that unnatural amino acids are included in MISTIC. We detected MISTIC labelled by a radioisotope by PSL (same method stated in <b>(iii)</b>). </p>

<p class="paragraph"> As we could see that MISTIC is not formed when unnatural amino acids are not added, we could determine that tRNA of amino acids which are inserted to the codon of amber competitively with RF1 does not exist. From this fact, we could confirm that unnatural amino acids are included in MISTIC including amber. </p> <br clear="right">

<p class="paragraph"> <b>(iv) Confirmation of the ability of access to biotin under the non-denatured condition</b> </p>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(b)-5. SDS-PAGE of PURE products</p>

</div>

<p class="paragraph"> By certifying whether or not MISTIC expressed in PURE frex are removed by the magnetic beads which streptavidin (SA) is connected, we confirmed that SA can access to niotin under the non-denatured condition. To exclude the influence of other PURE factors, we labelled it by 35S-Met and assayed by PSL. </p>

<p class="paragraph"> We loaded same amount of the product of PURE to all lanes. As only the amount of MISTIC with biotin is decreased, biotin connected to MISTIC is able to be accessed and MISTIC was removed from solution through that connection. </p> <br clear="right">

<p class="paragraph"> <b>(v) Confirmation of modification of MISTIC to cysteine</b>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(b)-6. SDS-PAGE of modified mistic (Cy3/SYPRO ORENGE)</p>

</div>

<p class="paragraph"> MISTIC was labelled by Cy3 maleimide (Cy3-MA) and confirmed whether cysteine residue of MISTIC is able to be modified or not. 0.45µL of 11mM Cy3-MA and 50µL of 3.26µM MISTIC were mixed and incubated for 30 minutes. Products were perdormed to electrophoresis using SDS-PAGE ageter purification and fluorescence was observed. After that, protein was dyed by SYPRO ORANGE and fluorescence was observed again. </p>

<p class="paragraph"> As we could see the fluorescence of Cy3 at the position of the band of MISTIC, the modification seemed to be successful. Comparing the intensity of fluorescence with other lanes which we put the same amount of Cy3-MA, the modification rate is presumed to be --%. </p>


<h1 class="big">1-1(c). Designing the Activator</h1> <p class="paragraph"> Two restriction enzymes, Hind&#8546; and Lambda Exonuclease, were compared to choose which is appropriate for the Activator. Two aspects were evaluated in this experiment: modification with oligonucleases and enzyme activity.</p>

<p class="paragraph"><b>(i) Evaluating modification with oligonucleatides</b></p>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(c)-1. BS(PEG)<sub>9</sub> for oligo-modification.</p>

</div>

<p class="paragraph">Hind&8546; and Lambda Exonuclease are modified with oligonucleotides to join the Activator and the Anchor. BS(PEG)<sub>9</sub> was used for the modification.</p>

<br clear="right">

<p class="paragraph">On Hind&#8546;, the modification was analyzed by Native-PAGE. Oligonucleotides are labelled with Cy3 and Hind&#8546; is labelled with SYBR GREEN to confirm the modification.</p>

<div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(c)-2. Cy3 fluorescence image of oligo-modified Hind&#8546; by Native-PAGE.</p>

</div>

<div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(c)-3. SYPRO-ORANGE fluorescence image of oligo-modified Hind&#8546; by Native-PAGE.</p>

</div>

<br clear="left">

<p class="paragraph">In the both figures, a band was observed in the same position in lane ○○. The modification of Hind3 with Cy3-oligonucleotides was therefore confirmed.</p>

<p class="paragraph">On Lamda Exonuclease, the modification was confirmed by Native-PAGE in the same way as Hind&#8546;.</p>

<div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(c)-4. Cy3 fluorescence image of oligo-modified Lamda Exonuclease by Native-PAGE.</p>

</div>

<div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(c)-5. SYPRO-ORANGE fluorescence image of oligo-modified Lamda Exonuclease by Native-PAGE.</p>

</div>

<br clear="left">

<p class="paragraph">The result shows Lamda Exonuclease was modified with oligonucleotide successfully. </p>

<p class="paragraph"><b>(ii) Evaluating enzyme activity</b></p> <div class="smallbox"> 

 <p class="smallimage"><img src=""></p>
 <p class="caption">Fig.1-1(c)-7. DNA cleavage reaction by Lamda Exonuclease.</p>

</div> <div class="smallbox"> 

 <p class="smallimage"><img src=""></p>
 <p class="caption">Fig.1-1(c)-6. DNA cleavage reaction by Hind&#8546;.</p>

</div> <p class="paragraph"> Hind&#8546; is an restriction endonuclease that recognizes base sequence 5’-AAGCTT-3’ and cleaves it, while Lambda Exonuclease degrades one strand from 5'-phosphoryl termini of dsDNA.</p>

<br clear="right"> <p class="paragraph"> In this experiment, enzyme activity of Hind&#8546; and Lambda Exonuclease was evaluated in various conditions. The purpose is to confirm that Hind&#8546; and Lambda Exonuclease have activity in condition for DNA origami. The activity was analyzed by Native-PAGE.</p>

<div class="smallbox"> 

 <p class="smallimage"><img src=""></p>
 <p class="caption">Fig.1-1(c)-9. Native-PAGE shows Lamda activity depending on PH.</p>

</div>

<div class="smallbox"> 

 <p class="smallimage"><img src=""></p>
 <p class="caption">Fig.1-1(c)-8. Native-PAGE shows Hind&#8546; activity depending on PH.</p>

</div>

<p class="paragraph"> First, enzyme activity depending on PH was analyzed.</p>

<p class="paragraph"> The result showed that Lambda Exonuclease can act in pH7.5-8.5. In pH9.0 and pH9.4, dsDNA seems to be unstable. So, pH7.5-8.5 is appropriate for reaction of Lambda Exonuclease and DNA. </p>

<br clear="right">

<div class="smallbox"> 

 <p class="smallimage"><img src=""></p>
 <p class="caption">Fig.1-1(c)-11. Native-PAGE shows Lamda activity depending on concentration of MgCl<sub>2</sub>.</p>

</div>

<div class="smallbox"> 

 <p class="smallimage"><img src=""></p>
 <p class="caption">Fig.1-1(c)-10. Native-PAGE shows Hind&#8546; activity depending on concentration of MgCl<sub>2</sub>.</p>

</div>

<p class="paragraph"> Then enzyme activity depending on concentration of MgCl<sub>2</sub> was analyzed.</p> <p class="paragraph"> The result showed that Lambda Exonuclease can act in mM MgCl<b>2</b>.</p>

<br clear="right">

<h1 class="sub">1-2. Embedding the Wall into the liposome</h1> <p class="paragraph"> Embedding the Wall into the liposome needs two steps: modifying the Wall with cholesterol and putting them into the liposome. The experiments were done individually. </p> <p class="paragraph"> (i) Hybridization of cholesterol oligomer with the Walls </p>

<p class="paragraph"> In this experiment, we examined the best concentration and the number of staples which the cholesterol oligomers can connect. Hybridization of cholesterol oligomer with the Walls is needed to penetrate the Walls into the membrane of liposomes. The result was assayed by 1% agarose gel electrophoresis. </p>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-2-1. Confocal microscope image of GUV including Walls.</p>

</div>

<p class="paragraph"> If the cholesterol oligomer was connected to the Walls the band would be smear. In sample 2_1~4, 3_1, 3_2, 5_1, 5_2, 6_1. Considering that sample 2 and 3 do not have staple for hybridization, the Walls of sample 2 and 3 aggregated by the existence of cholesterol oligomer. Sample 5_1, 5_2 and 6_1 are considered to be successful in hybridization. As the band of sample 5_2 and 6_1 are weaker than that of sample 5_1, we decided that the best condition was sample5_1. </p>


<br clear="right">

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-2-1. Confocal microscope image of GUV including Walls.</p>

</div>

<p class="paragraph"> In this experiment, the Wall which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV) to penetrate the liposome from inside. Inclusion of Motor-Monomers and Walls was observed by confocal microscope. </p>

<br clear="right">

The connection of Q-dot to the Wall was confirmed by agarose gel electrophoresis.

<div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-2-2. Agarose gel electrophoresis showing the existence of Q-dots.</p>

</div> <div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-2-3. Agarose gel electrophoresis showing the existence of DNA.</p>

</div> <br clear=left">

<p class="paragraph"> The first picture shows the existence of Q-dots and the second picture shows the existence of DNA. </p> <p class="paragraph"> Comparing the seventh, eighth and ninth lane from the left in the first picture, we can see the difference in the position of the Q-dots. Comparing the eighth and eighth lane from the left in the two pictures, we can see the position of DNA, in this case, Walls, and the Q-dots in the same position. This data shows that the Q-dots connected to the Walls through the connection of biotin and streptavidin. </p>


<h1 class="sub">1-3. Linking the Activator to the liposome</h1>

<h1 class="sub">1-4. Combining the Wall and the Activator</h1>

<h1 class="sub">1-5. Separating the Wall from the Activator</h1>

<a name="3"></a> <br> <br> <br> <h1 class="title"><a name="background">&nbsp;The Motor</a></h1>

<h1 class="sub">2-1. Producing the Motor-Monomer </h1>

<p class="paragraph"> Producing the Motor-Monomer is divided into three sections: Folding the Motor-Monomer body, synthesizing divalent streptavidin and equipping the Motor-Monomer body with divalent streptavidin. </p>

<h1 class="big">2-1(a). Folding the Motor-Monomer body </h1> <div class="smallbox"> <p class="smallimage"><img src="Time"></p> <p class="caption">Fig.2-1(a)-1. Gel analysis of the Monomers annealed in different time.</p> </div> <div class="smallbox"> <p class="smallimage"><img src="temperature"></p> <p class="caption">Fig.2-1(a)-2. Gel analysis of the Monomers annealed in different temperature.</p> </div>

<p class="paragraph">In this experiment, the assembly condition of the Motor-Monomer structure was optimized and results were analyzed by agarose gel electrophoresis. The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.</p>

<br clear="right">

<div class="smallbox"> <p class="smallimage"><img src="NaCl"></p> <p class="caption">Fig.2-1(a)-3. Gel analysis of the Monomers annealed in different concentration of NaCl.</p> </div> <div class="smallbox"> <p class="smallimage"><img src="MgCls"></p> <p class="caption">Fig.2-1(a)-4. Gel analysis of the Monomers annealed in different concentration of MgCl2.</p> </div>

<p class="paragraph">The optimum results were following: <ul style="margin:5px 35px;"> <li>Optimum concentration of MgCl<sub>2</sub> is 15 mM.</li>

<li>Optimum temperature of annealing is 45.3 &deg;C.</li>

<li>Optimum time of annealing is 5 hours.</li>

<li>Optimum concentration of NaCl is 2.5 mM.</li> </ul> </p> <br clear="right">

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1(a)-5. TEM image for the Motor-Monomers.</p>

</div>

<p class="paragraph"> The folding is corroborated by the TEM image. </p> <br clear="right">

<p class="paragraph">


<h1 class="big">2-1(b). Synthesizing divalent streptavidin</h1>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1(b)-1. SDS-PAGE showing purified divalent SA.</p>

</div> <p class="paragraph"> In this experiment, divalent streptavidin (SA) was synthesized for equipment to the Motor-Monomers. </p> <p class="paragraph"> TaKaRa Competent Cell BL21 was used for protein expression. The purification of divalent SA on the nickel-affinity column was analyzed by SDS-PAGE. </p> <br clear="right">

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1(b)-2. SDS-PAGE showing the affinity of divalent SA with biotin-modified oligos.</p>

</div> The affinity of divalent SA with biotin-modified oligonucleotides was then analyzed by SDS-PAGE. <br clear="right">

<h1 class="big">2-1(c). Equipping the Motor-Monomer with SA</h1> <p class="paragraph"> Because linking the Motor-Monomer and divalent SA uses modification of divalent SA by NHS ester reaction and click reaction, SA was linked with cy5-modified oligonucleotide via click reaction and NHS ester reaction, then the ability of modified SA to bind biotin-nucleotide was confirmed in this experiment.</p>


<div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1-3. .</p>

</div> <div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1-4. .</p>

</div> <br clear="left">

<p class="paragraph"> Figure 2-1-3 shows that SA was linked with cy5-modified oligonucleotide via click reaction. In Fig.2-1-4, because the lane of only Cy5 and NH2-modified oligonucleotide is not stained, the stained bands is from biotin-oligonucleotide. This picture shows modified SA can bind biotin. It may show the ability to bind biotin decreases as the amount of modification of SA increases, but the experiment of modification SA with Cy5-NHS deny. The reason of that may be derived from steric hindrance of Cy5-oligonucleotide which binds to SA.</p> <p class="paragraph"> These figures confirm the ability of modified SA to bind biotin-nucleotide.</p>


<h1 class="sub">2-2. Deactivating and activating the binding capacity of streptavidin </h1>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-2-1. Mechanism of this experiment.</p>

</div>

<p class="paragraph"> In this experiment, deactivation and reactivation of streptavidin (SA) were confirmed by Native PAGE. </p> <p class="paragraph">First, two complementary oligonucleotides having biotin or desthiobiotin were annealed. The oligos having biotin is named Linker and the oligos having desthiobiotin is named Blocker. The resulting dsDNA was then mixed with divalent SA for deactivation. After removing excess SA with biotin-beads, ds-DNA bound to divalent SA was cut by Hind&#8546; for reactivation. Another oligonucleotides having biotin, named Chaser, were then added to replace Blocker.</p>

<p class="paragraph">Blocker was labeled with Cy3 and Chaser was labeled with Cy5 to confirm the replacement by Native-PAGE.</p> <br clear="right">

<div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-2-2. Cy3 fluorescence image of Native-PAGE.</p>

</div> <div class="centerbox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-2-3. Cy5 fluorescence image of Native-PAGE .</p>

</div> <br clear="left">

<p class="paragraph"> In fig.2-2-3, multiple bands of SA-dsDNA complexes appears in lane 3 which is caused by non-ideal complexes. It is considered that biotin-beads successfully distinguished the ideal SA-dsDNA complex because the upper band in lane 3 mostly disappeared in lane 4.</p> <p class="paragraph"> In fig.2-2-4, a band at the same position of the ideal SA-dsDNA complex is only shown in lane 7. We therefore confirmed the replacement of Blocker with Chaser after cutting dsDNA by Hind&#8546; and that the replacement did not happen without Hind&#8546;.</p>

<h1 class="sub">2-3. Putting the Motor-Monomers into the liposome </h1>

<p class="paragraph"> In this experiment, Motor-Monomers which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV). Inclusion of Motor-Monomers was observed by confocal microscope. </p> <p class="paragraph"> GUVs were dyed by Nile Red. Yellow green dots indicate the fluorescence of Nile Red and red dots indicate the fluorescence of Q-dots combined with Motor-Monomers. </p>

<center> <div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-3-1. Confocal microscope image of GUV including Motor-Monomers.</p>

</div> <div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1-2. Confocal microscope image of GUV not including Motor-Monomers.</p>

</div> </center> <br clear="right">

<p class="paragraph"> Comparing the two pictures, we can see the red dots inside the GUV only in the picture showing the GUVs containing Motor-Monomers in the inner solution. From this fact, we could confirm the inclusion of Motor-Monomers into the GUV. As the excitation and fluorescence wave length of Nile Red and Q-dots were close, the membrane of GUV was shown in red even in the picture of GUV not including the Q-dots. </p> <br clear="right">

<p class="paragraph"> The connection of Q-dot to the Motor-Monomer was then confirmed by the agarose gel electrophoresis. </p>

<div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1-3. Agarose gel electrophoresis showing existence of Q-dots.</p>

</div> <div class="imagebox"> 

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1-3. Agarose gel electrophoresis showing existence of DNA.</p>

</div> <br clear="right">

<p class="paragraph"> The first picture shows the existence of Q-dots and the second picture shows the existence of DNA. Comparing the fourth, fifth and sixth lane from the left in the first picture, we can see the difference in the position of the Q-dots. Comparing the fifth and sixth lane from the left in the two pictures, we can see the position of DNA, in this case, Motor-Monomers, and the Q-dots in the same position. This data shows that the Q-dots connected to the Motor-Monomers through the connection of biotin and streptavidin. </p>


<br>


</body>

<footer style="position:relative; left:600px;"> <small> &copy; 2014 UTokyo Chem & Bio </small> </footer>

</html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2014/Kashiwa/Experiments&oldid=834228"