Biomod/2014/Kashiwa/Experiments: Difference between revisions

Revision as of 13:30, 24 October 2014

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>

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

</script>

// 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>

.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;}

}

column-content

{

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

} .firstHeading {

  display:none;
  width:0px;

}

globalWrapper

{

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

}

column-one

{

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

}

content

{

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

}

bodyContent

{

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

}

column-content

{

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

}

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>

 <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>

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 */

}

h1.big{

 font-size :18px;
 text-decoration: none;
 color: #000000;
 margin: 10px 15px;
 font-weight: bolder;
 }

h1.sub {

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

}

</style>

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

} // --></script>

  /* ■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"> Highlights</a></h1> <br>

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

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

  <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. Folding the Motor-Monomer </a></li>
           <li> <a href="#2-2">2-2. Deactivating and activating 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"> The Receptor</a></h1>

<h1 class="sub">1-1. Preparing the components </h1>

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

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(a)-1. Gel analysis of the walls annealed in different concentration of MgCl2.</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>

<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 °C.</li>

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

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

 <p class="image"><img src=""></p>
 <p class="caption">Fig.1-1(a)-2. TEM image of the Wall.</p>

</div>

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

<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> <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>TSEEKEQLSTAIDRMNEGLDAFIQLYNESEIDEPLIQLDDDTAELMKQARDMYGQEKLNEKLNTIIKQILSISVSEEGEKE<font color="red">HHHHHH</font></p> <p class="paragraph">+ is an abbreviation for unnatural amino acid.</p>

 <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>

 <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">

 <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>

 <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>

 <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>

 <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>

<h1 class="sub">1-2. Embedding the Wall into the liposome</h1>

 <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>

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

 <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="imagebox">

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

</div>

<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>

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

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

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

 <p class="image"><img src=""></p>
 <p class="caption">Fig.2-1-1. Gel analysis of the Motor-Monomer annealed in different concentration of MgCl2.</p>

</div>

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

<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 °C.</li>

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

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

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

</div>

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

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

<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>

 <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>

 <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>

</html>

Biomod/2014/Kashiwa/Experiments: Difference between revisions

Revision as of 13:30, 24 October 2014

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

research

Tools

@@ Line 317: / Line 317: @@
 <h1 class="sub">1-2. Embedding the Wall into the liposome</h1>
+<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="imagebox">
+  <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="imagebox">
+  <p class="image"><img src=""></p>
+  <p class="caption">Fig.1-2-3. Agarose gel electrophoresis showing the existence of DNA.</p>
+</div>
+<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>
@@ Line 370: / Line 399: @@
 <p class="paragraph">
-In this experiment, Motor-Monomers and Walls which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV). Inclusion of Motor-Monomers and Walls was observed by confocal microscope.
+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">