Biomod/2011/TeamJapan/Tokyo/Project/Results: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 199: Line 199:
|
|
:To construct the DNA tracks, we used microchannels to immobilize the substrate DNA at a specific region we designed. The construction of the microchannel is as follows. A mold of a microchannel was constructed with polyacetal resin by cutting the polystyrene resin as we designed with an endmill controlled by micro-machining system. The poly(dimethylsiloxane) (PDMS) and its hardener were mixed at the ratio of 10:1, and bubbles in the mixed PDMS solution was cleaned with a vacuum desiccator. The PDMS solution was poured over the mold and cured and hardened the PDMS solution at 75 degree Celsius for 1 hour. Then, the hardened PDMS was peeled off from the mold. The PDMS has a microchannel transferred from the mold.
:To construct the DNA tracks, we used microchannels to immobilize the substrate DNA at a specific region we designed. The construction of the microchannel is as follows. A mold of a microchannel was constructed with polyacetal resin by cutting the polystyrene resin as we designed with an endmill controlled by micro-machining system. The poly(dimethylsiloxane) (PDMS) and its hardener were mixed at the ratio of 10:1, and bubbles in the mixed PDMS solution was cleaned with a vacuum desiccator. The PDMS solution was poured over the mold and cured and hardened the PDMS solution at 75 degree Celsius for 1 hour. Then, the hardened PDMS was peeled off from the mold. The PDMS has a microchannel transferred from the mold.
|width="400px"|[[Image:Biomod2011 Team Tokyo 111030Biomod Construction of DNA track.png|center|Construction of DNA track|300px]]
 
|-
|
:Next, we constructed a DNA track using the PDMS microchannel. To make DNA tracks, we used microchannel and arrayed DNAs on a glass plate. To attach DNAs on a glass plate, we used disuccinimidyl suberate (DSS) as a linker between amino-modified DNA and an amino-modified glass plate (MAS-coated glass, Matsunami) [1]. The DSS linker reacts with amino groups that are exposed on the surface of the MAS-coated glass [2]. By the DSS linker, DNAs were attached on the glass plate by a covalent bond (Figure1). We poured an amino-modified DNA solution into the PDMS microchannel on the MAS-coated glass plate treated with DSS (Figure2); thus, the DNAs were arrayed as the shape of the microchannel we designed. We can design the shapes of microchannels freely, so we can make DNA tracks with arbitrary shapes. Finally, we observed the arrayed DNA track by hybridizing a fluorescence-labeled DNA complementary to the DNA strands of the DNA track (Figure3).
:Next, we constructed a DNA track using the PDMS microchannel. To make DNA tracks, we used microchannel and arrayed DNAs on a glass plate. To attach DNAs on a glass plate, we used disuccinimidyl suberate (DSS) as a linker between amino-modified DNA and an amino-modified glass plate (MAS-coated glass, Matsunami) [1]. The DSS linker reacts with amino groups that are exposed on the surface of the MAS-coated glass [2]. By the DSS linker, DNAs were attached on the glass plate by a covalent bond (Figure1). We poured an amino-modified DNA solution into the PDMS microchannel on the MAS-coated glass plate treated with DSS (Figure2); thus, the DNAs were arrayed as the shape of the microchannel we designed. We can design the shapes of microchannels freely, so we can make DNA tracks with arbitrary shapes. Finally, we observed the arrayed DNA track by hybridizing a fluorescence-labeled DNA complementary to the DNA strands of the DNA track (Figure3).
|width="400px"|[[Image:Tokyo tech：DNAimmobilization 5.png|center|A series of attaching aminated DNA to glass reaction|300px]]<br>
|width="400px"|[[Image:Biomod2011 Team Tokyo 111030Biomod Construction of DNA track.png|center|Construction of DNA track|300px]]<br>
[[Image:Tokyo tech：DNAimmobilization 5.png|center|A series of attaching aminated DNA to glass reaction|300px]]<br>
[[Image:Biomod2011 Team Tokyo 111030Biomod hybridization-Fl-DNA.png|Confirmed by DNA hybridization|center|300px]]
[[Image:Biomod2011 Team Tokyo 111030Biomod hybridization-Fl-DNA.png|Confirmed by DNA hybridization|center|300px]]
|}
|}

Revision as of 15:36, 2 November 2011


<html> <!--<head>--> <!-- We refer to the wiki of team iGEM Heidlberg 2009. --> 

      <style type="text/css">

/* ==================== 

  主に全体に関わるCSS

　　　==================== */ 

      body {behavior: url(http://www.xs4all.nl/~peterned/htc/csshover3-source.htc);}

      .clear {clear:both;}

body.mediawiki { 

                       font-size: 14px;

background-color:#707070; background-position: center center; background-attachment: fixed; background-repeat: no-repeat; font-family: Calibri, Verdana, helvetica, sans-serif; } 

               h1 {
                       padding: 0px 20px 5px 20px;
                       font-size: 34px;
                       font-weight: bold;
               }
               h2 {
                       padding: 20px 20px 5px 20px;
                       font-size: 25px;
                       color: #0083eb;
                       text-decoration: none;
                       font-weight: bold;}
                       border: none;
               h2 a {
                       color: #eb8300;
               }
               h3 {
                       padding: 20px 20px 5px 20px;
                       font-size: 20px;
                       color: #000;
                       font-decoration: none;
                       font-weight: bold;
               }
               h1.firstHeading {

display: none; 

               }

p { text-align: justify; } a:link { color: #00a5ea; text-decoration: none } a:visited { color:#00a5ea; text-decoration: none } a:hover { 

                       color: #eb8300;

text-decoration: none } a:active { color:#f29400; text-decoration: none } #bodyContent { 

                       width: 970px;
                       margin: 0px 0px 0px;
                       background-color:#ffffff;
                       border-width: 0px 1px 0px 1px;
                       border-color: #000000;
               }
               #content {
                       padding-left: 0px; width: 970px;
               }

table#team_members { 

                       text-align: justify;
                       border: 0;
               }

table#team_members h2, table#team_members h3 { 

                       clear: both;
               }

#content * a:hover { 

                       text-decoration:none;
               }

#main_wrapper { 

                       position:absolute;
                       left:0px;
                       top:20px;

margin-top: 0; width: 969px; height: 221px; align: center; border-style: solid; 

                       border-color: white;

} /* ==================== 

  メニューの画像を変更できる部分
  ==================== */

#header { 

                       position:relative;
                       left:0px;
                       top:0px;

margin-top: 0; width: 969px; height: 221px; align: left; background-color: #FFFFFF; 

                       background-image: url(http://openwetware.org/images/e/e0/NEW_header.jpg);

} /* ==================== 

  以下、特殊なclassに適用される
  ==================== */

#navigation { position:absolute; 

                       left:18px; top:155px; width:1200px; height:69px;

z-index:100; 

                       background-color: transparent;
                       float: left;

color: #0000FF; } #super_main_wrapper { 

                       position:absolute;
                       left:0px;
                       top:227px;

width: 975px; align: center; background-color: #ede8e2; 

                       heigth: auto;
               }
               #SubWrapper {
                       width: 645px;
                       padding: 0px;
                       border-left:4px solid #ede8e2;
                       float: left;
                       margin-top: 0px;
                       background-color: #ede8e2;
               }
               #SubWrapper * p, #SubWrapper p  {
                       padding: 0 20px;
                       text-align: justify;
                       font-size: 12px;
               }
               #SubWrapper * h3, #SubWrapper h3 {
                       padding-top: 10px;
                       font-size: 18px;
               }
               #news {
                       width: 322px;
                       margin-top: 0px;
                       float: left;
                       background-color: #d8d5d0;
                       border-right:4px solid #ede8e2;
               }
               #news p {
                       padding: 0 20px 20px 20px;
                       text-align: justify;
                       font-size: 12px;
               }
               #news h3 {
                       padding: 10px 20px;
                       font-size: 18px;
               }
               #mission_box {
                       width:650px;
                       float: left;
               }
               #team_box, #heartbeat_box, #notebook_box, #parts_box, #gallery_box, #sponsors_box, .boxy {
                       width:215px;
                       float: left;
                       padding: 10px 0 0 0;
               }
               div.tleft {
                       border-width: 0px;
                       margin:0;
                       padding:0;
                       border-color:transparent;
               }

/* ==================== 

  ここからプルダウン周辺のデザイン
  ==================== */
  1. menu * {

margin: 0; padding: 0; }

  1. menu {

behavior: url(http://www.xs4all.nl/~peterned/htc/csshover3-source.htc); <!-- fuer ie6 --> font-family: calibri, verdana, sans-serif; 

       font-color: #ffffff;

font-size: 19px; background-color: transparent; float:left; padding: 12px 0 0 0; }

  1. menu ul {

float: left; list-style: none; }

  1. menu ul li {

background-color:transparent; 

       position:relative;

float:left; list-style: none; padding: 10px 20px 0 0; 

       font-weight: bold;
       width: auto;

}

  1. menu a {

color: #FFFFFF; display: inline; text-decoration: none; }

  1. menu a:visited {

color:#FFFFFF; text-decoration: none }

  1. menu a:hover {

color: #00a5ea; }

  1. menu ul li ul {

display: none; position: absolute; left: 0px; 

       width: 155px;
       heigth: 1%;

font-size: 19px; opacity: 0.8; list-style: none; 

       top: 30px;
       padding-top: 20px;
       z-index:500;

}

  1. menu ul li:hover ul {

display: inline; 

       background-position: bottom;

}

  1. menu ul li ul li {

width: 100%; list-style: none; 

       background-color: #000;
       margin: -1px;
       padding: 0px 0 0 5px;
       display: inline;

}

</style> <!-- </head> -->


<!-- ############################# BODY!!!!! #################################### -->

<!-- <body> --> <!--[if IE]> <style type="text/css">

  1. SubWrapper {width: 645px;}
  2. news {width: 317px;}
  3. super_main_wrapper {position:static;}
  4. navigation {left: 15px;}
  5. menu ul li ul {left: -2px; top: 22px;}

</style> <![endif]--> <div id="main_wrapper"> 

 <div id="header">

<div id="navigation"> <div id="menu" style="position:static"> <ul> <li><a class="aMain" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo">Home</a></li> <li><a class="aTeam" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Team/Students">Team</a></li> <li><a class="aProject" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Project</a> <!-- <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Overview</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/introduction">Introduction</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Model">Model</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Devices">Devices</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Modes">Modes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> </ul> --> <li><font color="#ffffff">Results</font> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Experiments</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Design</a></li> </ul></li> <!-- <li><a class="Simulation" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a class="DNA design" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Designs</a></li> --> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook">Notes</a></li> <li><a class="aNotebook" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sponsors/">Sponsors</a></li> <li><a class="aSitemap" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sitemap">Sitemap</a></li>

</ul> </div><!-- end drop menu --> </div> <!-- end navigation --> </div> </div> <!-- For IE 6 --> <script type="text/javascript">

/***********************************************

  • CSS Vertical List Menu- by JavaScript Kit (www.javascriptkit.com)
  • Menu interface credits: http://www.dynamicdrive.com/style/csslibrary/item/glossy-vertical-menu/
  • This notice must stay intact for usage
  • Visit JavaScript Kit at http://www.javascriptkit.com/ for this script and 100s more
                                                                                              • /

var menuids=new Array("verticalmenu") //Enter id(s) of UL menus, separated by commas var submenuoffset=-2 //Offset of submenus from main menu. Default is -2 pixels.

function createcssmenu(){ for (var i=0; i<menuids.length; i++){ 

 var ultags=document.getElementById(menuids[i]).getElementsByTagName("ul")
   for (var t=0; t<ultags.length; t++){
   var spanref=document.createElement("span")

spanref.className="arrowdiv" spanref.innerHTML="&nbsp;&nbsp;" ultags[t].parentNode.getElementsByTagName("a")[0].appendChild(spanref) 

   ultags[t].parentNode.onmouseover=function(){
   this.getElementsByTagName("ul")[0].style.left=this.parentNode.offsetWidth+submenuoffset+"px"
   this.getElementsByTagName("ul")[0].style.display="block"
   }
   ultags[t].parentNode.onmouseout=function(){
   this.getElementsByTagName("ul")[0].style.display="none"
   }
   }
 }

} if (window.addEventListener) window.addEventListener("load", createcssmenu, false) else if (window.attachEvent) window.attachEvent("onload", createcssmenu) </script> <!-- </body> --> </html>













Experimental Results

<html><body> <td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#The_body_of_the_DNA_ciliate"><img src="http://openwetware.org/images/4/4a/BIOMOD_Tokyo20111031Result_figure_ciliate.png" border=0 width=200 height=200></a></td> </body></html>
  • We constructed the micrometer-sized body of the DNA ciliate by surface modification. Shown in detail below
<html><body> <td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#1._Free_moving_mode"><img src="http://openwetware.org/images/a/ac/BIOMOD_Tokyo20111031Result_figure1.png" border=0 width=200 height=200></a></td> <td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#2._The_track_walking_mode"><img src="http://openwetware.org/images/0/05/BIOMOD_Tokyo20111031Result_figure2.png" border=0 width=200 height=200></a></td> <td align="center" width="200px"><a href="/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results#3._Light-irradiated_gathering_mode"><img src="http://openwetware.org/images/b/bf/BIOMOD_Tokyo20111031Result_figure3.png" border=0 width=200 height=200></a></td> </body></html>
  • We confirmed that DNA ciliate can move freely and randomly alomost all area based on Brownian motion. Shown in detail below.
  • We confirmed that substrate DNA are cut by deoxyribozyme legs of DNA ciliate.
  • We constructed DNA tracks by using the microchannel.
  • We confirmed that DNA ciliates move directionally in simulation.
    Shown in detail below
  • We designed the DNA which react.
  • When we irradiated UV, we succeeded to gather beads at the spot where we irradiate UV.
    Shown in detail below.

The construction of the body of the DNA ciliate
The DNA ciliate has a micrometer-sized body with DNAs as cilia. We constructed the micrometer-sized body of the DNA ciliate using a polystyrene bead. The DNAs as cilia on the body was made of deoxyribozyme DNAs. To attach the deoxyribozyme DNAs to the polystyrene body, a chemical covalent bond, the amide bond, was utilized. Here, we show the construction method of the DNA ciliate and the investigation of the constructed DNA ciliate based on deoxyribozyme activity assays.

Method

  • Two experiments were needed to complete developing DNA ciliate body.
    First experiment was creating DNA ciliate by attaching DNAs to polystyrene beads. This process is used the reaction of connecting amino group of aminated DNAs and carboxylic acid of polystyrene beads. We took two Method to react. Both Method are used the common reaction, but chemical materials are different.
The method is used EDAC. EDAC reacts with both aminated DNAs and polystyrene beads’ carboxylic acid.
Figure1.the result of PAGE of φ200 nm polystyrene beads using NHS and EDC.

Second experiment is confirming whether deoxyribozyme is attached to polystyrene beads and able to cleave substrate. We confirm deoxyribozyme activity by urea-PAGE. Making mixture of DNA ciliate and substrate and Zn2+ ions. If DNA ciliate has deoxyribozyme activity, substrate is cleaved and the band of cleaved substrate appears as a band.
  • Creating DNA ciliate, we use (1) and (2) protocols.
    • (1) The method of using EDC and NHS is here.
    • (2) The method of using EDAC…
  • Confirming DNA ciliate, we use (3) and (4) protocols
    • (3) The method of electrophoresis is here.
    • (4) The method of making sample is


The results of electrophoresis of DNA ciliate by using EDAC

In these experimentations, we could confirm two things. First was the deoxyribozyme activity of DNA ciliate body. Second was the firmness of the bond between polystyrene beads and deoxyribozyme. The deoxyribozyme activity of DNA ciliate body can be confirmed. If there is deoxyribozyme activity of DNA ciliate body, the cleaved substrate band is appeared. Furthermore, DNA ciliate body is removed before loading to polyacrylamide gel by centrifuge, so if deoxyribozyme can’t be attached to polystyrene beads firmly, the leg band appeared. In all pictures of gels, there are cleaved substrate bands in lane 8, so it is confirmed that all polystyrene beads are attached deoxyribozyme. Furthermore, there are not deoxyribozyme bands in lane 8, so it is confirmed that all polystyrene beads are attached deoxyribozyme firmly and the deoxyribozyme activity is not because of dislocated deoxyribozyme. In conclusion, we succeeded in making DNA ciliate body.

Comparing four results, about both 200 nm and 1um polystyrene beads in diameter, the cleaved substrate band of EDAC method is stronger than EDC method, so DNA ciliate body made by EDAC method is better than EDC method. We decided using DNA ciliate body bodymade by EDAC method.
The result of denaturing PAGE of φ200 nm polystyrene beads using EDAC.
*The result of denaturing PAGE of φ1 μm polystyrene beads using EDAC.


Lanes of 5 to 8 are needed for checking deoxyribozyme activity of DNA ciliate body. Lane 5 and 6 are lanes for checking to polystyrene beads. If polystyrene beads had deoxyribozyme activity, the cleaved band would be appeared. Lanes of 7 and 8 are needed for checking that DNA ciliates body have deoxyribozyme activity. If DNA ciliate has normal deoxyribozyme activity, the cleaved band is appeared in lane 8 because metal ions are needed for deoxyribribozyme activity.

Three independent modes of the DNA ciliate

1. Free moving mode
In the free moving mode, the DNA ciliates freely and randomly move around in a broad range of space. Here, we confirmed the free moving mode by the observation of the DNA ciliate that exhibited the Brownian motion in an aqueous solution. (see below)

Method

In the observation of the DNA ciliate, we used 1× saline-sodium citrate (SSC) buffer with 3% bovine serum albumin (BSA) (the materials for experiments are listed in Protocols). The sizes of DNA ciliate bodies were 200 nm and 1 μm. We put the solution including the DNA ciliates on a glass slide and covered by a cover slip. The solutions including the DNA ciliates were observed by a phase-contrast microscope and took videos (the equipment for experiments is also listed in Protocols).

Results

Video (A1) (the left two videos) shows the observation results of the DNA ciliates with a diameter of 200 nm under the optical microscope. Video (B1) (the right two videos) shows the observation results of the DNA ciliates with a diameter of 1 μm. The lower videos are enlarged views of the upper videos.

<html><body> <table rules="all" frame="void" width="100%" cellspacing="0"> 

<tr align="center">
 <td>Video (A1)

<iframe width="450" height="259" src="http://www.youtube.com/embed/uGRn9Z8inW4?rel=0" frameborder="0" allowfullscreen></iframe></td> 

 <td>Video (B1)

<iframe width="450" height="259" src="http://www.youtube.com/embed/-zzB6UeWKoM?hl=ja&fs=1" frameborder="0" allowfullscreen></iframe></td> 

</tr>
<tr align="center">
 <td>Video (A2)

<iframe width="450" height="259" src="http://www.youtube.com/embed/E1vW6eaABcQ" frameborder="0" allowfullscreen></iframe></td> 

 <td>Video (B2)

<iframe width="450" height="259" src="http://www.youtube.com/embed/jKpgMfls3Kw" frameborder="0" allowfullscreen></iframe></td> 

</tr>

</table> </body></html>

From these videos, we observed that the DNA ciliates were freely and randomly moving in the solution. By comparing the motion of the DNA ciliates with a diameter of 200 nm and 1 μm, the smaller DNA ciliates (200 nm in diameter) moved more strongly than the larger DNA ciliates (1 μm in diameter). We observed a very slow directional flow of the solution as an experimental artifact but the motion of the DNA ciliate was random independently of the flow. In addition, we observed some DNA ciliates that did not move at all; the DNA ciliates were probably crystalized one another or sticking at the surface of the glass slide.
In conclusion, we achieved the free moving mode of the DNA ciliate. The motion of the DNA ciliate was based on the Brownian motion of the DNA ciliates. The random motion of the smaller DNA ciliates was stronger than that of the larger DNA ciliate. This result consistent with the theory of the Brownian motion described in Project page.

2. The track walking mode
The DNA ciliates walk directionally along the DNA tracks using deoxyribozyme cleving activity as described in Project page. We designed a deoxyribozyme and a substrate DNA for the track (see DNA design).
As already mentioned in the project page, we used "Deoxyribozyme-substrate reaction "
Here, we show the results of (1) conformation of deoxyribozyme activity, (2) construction of DNA tracks, and (3) investigation of the directional walking of the DNA ciliate.

Confirmation of Deoxyribozyme activity

We confirmed the cleaving activity of the deoxyribozyme for cilia attached on the DNA ciliate body using the polyacrylamide gel electrophoresis. Actually, the results have already been shown in the section of the confirmation of constructing the DNA ciliate body above (see the results) From the results, we conclude that the deoxyribozyme activity for the substrate DNA worked as we designed.

Construction of DNA tracks

Method of the construction of DNA tracks

To construct the DNA tracks, we used microchannels to immobilize the substrate DNA at a specific region we designed. The construction of the microchannel is as follows. A mold of a microchannel was constructed with polyacetal resin by cutting the polystyrene resin as we designed with an endmill controlled by micro-machining system. The poly(dimethylsiloxane) (PDMS) and its hardener were mixed at the ratio of 10:1, and bubbles in the mixed PDMS solution was cleaned with a vacuum desiccator. The PDMS solution was poured over the mold and cured and hardened the PDMS solution at 75 degree Celsius for 1 hour. Then, the hardened PDMS was peeled off from the mold. The PDMS has a microchannel transferred from the mold.
Next, we constructed a DNA track using the PDMS microchannel. To make DNA tracks, we used microchannel and arrayed DNAs on a glass plate. To attach DNAs on a glass plate, we used disuccinimidyl suberate (DSS) as a linker between amino-modified DNA and an amino-modified glass plate (MAS-coated glass, Matsunami) [1]. The DSS linker reacts with amino groups that are exposed on the surface of the MAS-coated glass [2]. By the DSS linker, DNAs were attached on the glass plate by a covalent bond (Figure1). We poured an amino-modified DNA solution into the PDMS microchannel on the MAS-coated glass plate treated with DSS (Figure2); thus, the DNAs were arrayed as the shape of the microchannel we designed. We can design the shapes of microchannels freely, so we can make DNA tracks with arbitrary shapes. Finally, we observed the arrayed DNA track by hybridizing a fluorescence-labeled DNA complementary to the DNA strands of the DNA track (Figure3).
Construction of DNA track
Construction of DNA track

A series of attaching aminated DNA to glass reaction
A series of attaching aminated DNA to glass reaction

Confirmed by DNA hybridization
Confirmed by DNA hybridization

Results of the construction of DNA tracks

Figure 4 shows a picture of the polyacetal resin mold (the mold for microchannels are shown by broken lines). This is a part of large microchannels we used to array DNAs. Figure 5 shows a fluorescence microscope image of the DNA tracks. In Figure 5, we observed the fluorescence of the DNA hybridizing with the DNA track arrayed on the glass plate. In addition, Figure 6 shows the whole picture of the large DNA tracks with a human like shape (observed similarly).
This figure is microchannel in PDMS-mold. This figure was observed by phase contrast.
This figure is the result of arraying DNAs on glass plate using microchannel of Figure4 and hybridized with their complementary fluorescent labeling DNA strands. This figure was observed by fluorescent phase contrast.
The microchannel of human form The left image is the design drawing. The right figure is the result of hybridization. Because camera view was too narrow to observe total image, we stuck together the part of pictures.
In conclusion, we successfully constructed DNA tracks using microchannels, and we confirmed the ability of hybridization between the immobilized DNA as the tracks and its complementary DNA. Thus, we believe that the deoxyribozyme on the DNA ciliate also hybridizes with the track DNA, which has a complementary DNA sequence of the deoxyribozyme.

Investigation of the directional walking by simulations

We investigated the directional walking of the DNA ciliate by numerical simulations (see Simulations)

3. Light-irradiated gathering mode
In the light-irradiated gathering mode, DNA ciliates gather at a specific area responding to UV irradiation. This mode is achieved by UV-switching DNA devices and gathering of DNA ciliates.
In this page, we show the two experimental results: confirmation of UV-switching and observation of gathering DNA ciliates. It is confirmed that UV-switching system worked and that DNA ciliates gathered, so the light-irradiated gathering mode will be achieved.

UV-switching system

The UV-switching DNA has a stem-loop structure and short blocking DNA, which blocks hybridization of deoxyribozyme. After UV irradiation, this loop becomes open, and hybridize with the deoxyribozyme. (more detail...)


1. Confirmation of UV-switching

Results

Image of non-denaturing 20% PAGE for the confirmation of UV-switching
  • Non-denaturing 20% PAGE result is here.
U…UV-switching-trap-DNA
B…Blocking DNA
D…Deoxyribozyme DNA
Reaction solution…A 0.225uM and B 0.45uM and D 0.225uM
All solutions are in 5x SSC (sodium citrate 75mM)
  • From left, these bands mean followings.
  1. U 0.225uM and Mg2+ 80mM
  2. B 0.45uM and Mg2+ 80mM
  3. D 0.225uM and Mg2+ 80mM
  4. U 0.225uM and B 0.45uM and Mg2+ 80mM
  5. U 0.225uM and D 0.225uM and Mg2+ 80mM (UV isn’t spotted)
  6. U 0.225uM and D 0.225uM and Mg2+ 80mM (UV is spotted for 60 min.)
  7. Reaction solution (UV isn’t spotted)
  8. Reaction solution (UV is spotted for 15 min.)
  9. Reaction solution (UV is spotted for 60 min.)
  10. Reaction solution and Mg2+ 80mM (UV isn’t spotted)
  11. Reaction solution and Mg2+ 80mM (UV is spotted for 15 min.)
  12. Reaction solution and Mg2+ 80mM (UV is spotted for 60 min.)
  • The control bands were appeared in lane 1 to 6. Lane 4 (U 0.225uM and B 0.45uM and Mg2+ 80mM) means the bands when the loop is stable and hybridization U and B (band U-B). Lane 5 (U 0.225uM and D 0.225uM and Mg2+ 80mM (UV isn’t spotted)) means the bands when the loop is open and hybridization of U and D. Lane 6 (U 0.225uM and D 0.225uM and Mg2+ 80mM (UV is spotted for 60 min.)) means the bands when the loop is open and spotted UV (band U-D).
  • In the presence of Mg2+, the switching was caused clearly (lane 10 to 12) because of the stable effect of Mg2+.
  • Before UV irradiation (lane 10), the UV-switching DNA was closed state (band U-B). After UV irradiation (lane 11, 12), the band shifted to the position of hybridized state (band U-D). Thus, the UV-switching device we designed worked successfully as we intended.

2. Gathering at the specific area

A fluorescent image of DNA ciliate gathering at the specific area

method

  • Attaching complementary DNA of deoxyriboazyme on a glass plate
  • Making the situation which deoxyribozymes hybridize with complementary DNA on the glass plate
How to make the situation for hybridization is here
  • Putting DNA ciliates on the glass plate
  • Waiting for 2 hours
  • Observing the DNA ciliates under an fluorescent microscope

result

  • A fluorescent image of the DNA ciliates gathering at the spot of complementary DNA is here.
  • Complementary DNA was attached on upper-right area in this image.
There was no DNA in lower-left area in this image.
  • DNA ciliates gathered at the spot of complementary DNA, and didn't gather at another area. Following this result, it was confirmed that DNA ciliates can gather at the specific area after UV irradiation.
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2011/TeamJapan/Tokyo/Project/Results&oldid=562221"