Team:Tokyo Tech

From 2008.igem.org

(Difference between revisions)
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<div class=Section1 style='layout-grid:18.0pt'>
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US
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<p class=MsoNormal><b><u><font size=4>Our project</font></u></b></p>
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style='font-size:14.0pt'>Our project<o:p></o:p></span></u></b></p>
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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          <td class="a" width="0%">　</td>
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          <td class="a">
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<p class=MsoNormal><b><u><font size=4>We want to make a <i style='mso-bidi-font-style:normal'>Coli Touch!!</font></u></b></p>
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<td class="a" width="3%">　</td>
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<p class=MsoNormal style='text-indent:5.25pt;mso-char-indent-count:.5'><span
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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lang=EN-US>We want to make a <i style='mso-bidi-font-style:normal'>Coli Touch!!</i></span></p>
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        <tr>  
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          <td class="a" width="0%">　</td>
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<p class=MsoNormal><span style='font-family:"ＭＳ 明朝","serif";mso-ascii-font-family:
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          <td class="a">
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Century;mso-hansi-font-family:Century'>→</span><span lang=EN-US>We try to construct a bacterial ‘touch panel’ which is colored by the pressure.
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<p class=MsoNormal>We try to construct a bacterial ‘touch panel’ which is colored by the pressure.</p></td>
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</span></p>
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<td class="a" width="3%">　</td>
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        </tr>
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      </table>
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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<p class=MsoNormal style='text-indent:10.5pt;mso-char-indent-count:1.0'><span
<p class=MsoNormal style='text-indent:10.5pt;mso-char-indent-count:1.0'><span
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lang=EN-US>We name it <i>Coli touch.</i></span></p>
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lang=EN-US>We name it <i>Coli touch.</i></span></p></td>
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<td class="a" width="3%">　</td>
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molecule input occur concentration gradient, light input depend on location of a
molecule input occur concentration gradient, light input depend on location of a
container. So, we thought input style except these. That is “press”. Press input
container. So, we thought input style except these. That is “press”. Press input
-
has an advantage that input signal travel uniformly in a container.</i></span></p>
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has an advantage that input signal travel uniformly in a container.</span></p></td>
<td class="a" width="3%">　</td>
<td class="a" width="3%">　</td>
         </tr>
         </tr>
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<! Why pressure?>
 
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US
 
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style='font-size:14.0pt'>Why pressure?<o:p></o:p></span></u></b></p>
 
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<p class=MsoNormal style='text-indent:5.25pt;mso-char-indent-count:.5'><span
 
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lang=EN-US>Example of input style to genetic circuit</span></p>
 
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<p class=MsoNormal style='text-indent:10.5pt;mso-char-indent-count:1.0'><span
 
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lang=EN-US>- Heat: thermal gradient </span></p>
 
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<p class=MsoNormal style='text-indent:10.5pt;mso-char-indent-count:1.0'><span
 
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lang=EN-US>- Small molecule: concentration gradient</span></p>
 
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<p class=MsoNormal style='text-indent:10.5pt;mso-char-indent-count:1.0'><span
 
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lang=EN-US>- Light: strength depend on location of container</span></p>
 
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<p class=MsoNormal><span lang=EN-US><span
 
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style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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</span></span></p>
 
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<p class=MsoNormal><span lang=EN-US><span
 
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style='mso-spacerun:yes'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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<IMG src="https://static.igem.org/mediawiki/2008/b/be/Tech_%E7%9F%A2%E5%8D%B0.jpg" align="middle">&nbsp;&nbsp;</span>Difficulty in uniform introduction</span></p>
 
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<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
 
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<p class=MsoNormal><span lang=EN-US><span style='mso-spacerun:yes'>&nbsp;
 
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</span><span style='color:red'>Press!</span><span
 
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style='mso-spacerun:yes'>&nbsp; </span>Confirmatory experiment </span></p>
 
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<p class=MsoNormal><span lang=EN-US>Prospect of technological application!! </span></p>
 
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<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
 
<! ---------------------------------------------------------------------------------------------------------->
<! ---------------------------------------------------------------------------------------------------------->
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US>Pressure
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<p class=MsoNormal><b><u><font size=4>Mechanism</font></u></b></p>
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inducible promoter - P<sub>lac </sub><o:p></o:p></span></u></b></p>
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<p class=MsoNormal style='text-indent:5.25pt;mso-char-indent-count:.5'><span
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lang=EN-US>The affinity of LacI for the lac operator is known to decrease due
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lang=EN-US>We construct genetic circuits that can respond to pressure signal. Lac promoter was known as a pressure inducible promoter. Lac promoter is repressed by LacI protein. The repression is released by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside). Besides IPTG induction, pressure induction is possible. Under high pressure, the affinity of lac protein for lac operon decreases by transition of Lac protein from tetramer to dimer. Through this mechanism, Lac promoter is induced by pressure. </span></p></td>
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to a tetramer to dimer transition of LacI </span></p>
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<td class="a" width="3%">　</td>
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        </tr>
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<IMG src="https://static.igem.org/mediawiki/2008/b/bd/Test.jpg" alt="[サンプルの絵]" width="300">
<IMG src="https://static.igem.org/mediawiki/2008/b/bd/Test.jpg" alt="[サンプルの絵]" width="300">
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<! strategy>
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<p class=MsoNormal><b><u><font size=4>Strategy</font></u></b></p>
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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style='font-size:14.0pt'>Strategy<o:p></o:p></span></u></b></p>
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          <td class="a" width="0%">　</td>
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          <td class="a">
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<p class=MsoNormal>We thought two projects, basic project and application project. Basic project consist of two categories. One is construction of pressure responsive genetic (1). The other is creating basic device for a touch display (2). Application project also consist of two categories. One is development of low pressure inducible promoter (3). The other is construction of pressure responsive toggle switch (4). In the future, we want to create the touch display made of E. coli.  </p></td>
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          <td class="a" width="3%">　</td>
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        </tr>
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<IMG src="https://static.igem.org/mediawiki/2008/c/cf/Test2.jpg" alt="[サンプルの絵]" width="700">
<IMG src="https://static.igem.org/mediawiki/2008/c/cf/Test2.jpg" alt="[サンプルの絵]" width="700">
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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<! ---------------------------------------------------------------------------------------------------------->
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<! Equipment. Instrument for pressure experiment>
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          <td class="a" width="0%">　</td>
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          <td class="a">
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<p class=MsoNormal>We used these equipments (show figure) for pressure experiments. </p></td>
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US
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          <td class="a" width="3%">　</td>
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style='font-size:14.0pt'>Equipment. Instrument for pressure experiment<o:p></o:p></span></u></b></p>
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        </tr>
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<IMG src="https://static.igem.org/mediawiki/2008/a/a3/Tech_kaatu.jpg" align="middle" width="500">
<IMG src="https://static.igem.org/mediawiki/2008/a/a3/Tech_kaatu.jpg" align="middle" width="500">
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<small><p class=MsoNormal style='text-indent:5.25pt;mso-char-indent-count:.5'><span
 
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lang=EN-US>Fig 4: Fig4-1 is a pressure vessel. Fig4-4 is a pressurizing system.
 
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Fig4-2 show a inside of the vessel. The inside is filled with water and the
 
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withstand pressure container is put into the inside. Fig4-3 show a water bath
 
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placed some vessel.</span></p></small>
 
<! ---------------------------------------------------------------------------------------------------------->
<! ---------------------------------------------------------------------------------------------------------->
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<! 1.Construction>
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<p class=MsoNormal><b><u><font size=4>1.Construction</font></u></b></p>
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US>Strategy
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1.Construction<o:p></o:p></span></u></b></p>
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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          <td class="a" width="0%">　</td>
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<p class=MsoNormal>We construct these plasmids (show figure). </p></td>
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          <td class="a" width="3%">　</td>
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        </tr>
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<IMG src="https://static.igem.org/mediawiki/2008/6/63/Tech_constraction.jpg" align="middle">
<IMG src="https://static.igem.org/mediawiki/2008/6/63/Tech_constraction.jpg" align="middle">
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US>Confirmatory
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experiment<o:p></o:p></span></b></p>
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<IMG src="https://static.igem.org/mediawiki/2008/6/6b/Tech_plac_g1.jpg" align="middle">
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          <td class="a">
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<small><p class=MsoNormal style='text-indent:5.25pt;mso-char-indent-count:.5'><span
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<p class=MsoNormal>Each part is constructed on low-copy plasmid “pSB6”. </p></td>
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lang=EN-US>Fig1:Pressure-response of Plac-GFP under several pressures measured by flow cytometer</span></p></small>
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          <td class="a" width="3%">　</td>
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<! ---------------------------------------------------------------------------------------------------------->
<! ---------------------------------------------------------------------------------------------------------->
 +
<p class=MsoNormal><b><u><font size=4>2. Basic device</font></u></b></p>
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<IMG src="https://static.igem.org/mediawiki/2008/e/e5/Tech_pic_30.jpg" >&nbsp;<IMG src="https://static.igem.org/mediawiki/2008/a/a2/Tech_pic_rp.jpg" height="241">&nbsp;<IMG src="https://static.igem.org/mediawiki/2008/f/f6/Tech_pic_nega.jpg" height="241">
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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<small><p class=MsoNormal style='text-indent:5.25pt;mso-char-indent-count:.5'><span
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          <td class="a" width="0%">　</td>
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lang=EN-US>Fig2-1:Plac-GFP under 30MPa&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig2-2:Plac-GFP under 0.1MPa&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig2-3:⊿-GFP under 30MPa</span></p></small>
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          <td class="a">
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<p class=MsoNormal>We created pressurizing device for first step of creating touch display. This device has two holes (show figure). Each hole contains tubes and water. Inside tubes E. coli is cultivated. Pressure can travel to inside tubes. One hole (B) is covered with a block made of an acrylic glass. (show figure) Therefore the hole is not pressurized by water. The other (A) is covered with a plastic tape (show figure). Therefore the hole is pressurized.                        </p></td>
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<! ---------------------------------------------------------------------------------------------------------->
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          <td class="a" width="3%">　</td>
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US
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style='font-size:14.0pt'>Stratagy 2. Basic device</span></u></b></p>
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      </table>
<IMG src="https://static.igem.org/mediawiki/2008/9/9d/Tech_youki.jpg" align="middle" width="500" >
<IMG src="https://static.igem.org/mediawiki/2008/9/9d/Tech_youki.jpg" align="middle" width="500" >
<! --------------------------------------------------------------------------------------------------------->
<! --------------------------------------------------------------------------------------------------------->
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US
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<p class=MsoNormal><b><u><font size=4>3. Development of low pressure inducible promoter </font></u></b></p>
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style='font-size:14.0pt'>Stratagy 3. Development of low pressure inducible
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promoter <o:p></o:p></span></u></b></p>
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<p class=MsoNormal><span lang=EN-US>We performed random mutagenesis to lac
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<table width="100%" border="0" cellspacing="0" cellpadding="0">
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promoter and tried to screen promoters with FACS.</span></p>
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          <td class="a" width="0%">　</td>
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<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
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          <td class="a">
-
 
+
<p class=MsoNormal>In the stage of basic project, very high pressure (30MPa) is required for pressure induction of lac promoter. But lower pressure response is needed for creating touch display. Therefore we develop lower pressure inducible promoter. We performed random mutagenesis to lac promoter and tried to screen promoters with FACS.</p></td>
-
<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
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          <td class="a" width="3%">　</td>
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<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
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<IMG src="https://static.igem.org/mediawiki/2008/3/3e/Tech_dev1.jpg" align="middle" width="500" >
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<p class=MsoNormal><span lang=EN-US>Strategy for Isolation of promoters </span></p>
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<! --------------------------------------------------------------------------------------------------------->
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<p class=MsoNormal><b><u><font size=4>4.Genetic toggle switch to implement rewritable function </font></u></b></p>
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<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US>Future
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work<o:p></o:p></span></b></p>
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<p class=MsoNormal style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2;
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tab-stops:list 36.0pt'><![if !supportLists]><span lang=EN-US style='font-family:
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"Arial","sans-serif";mso-fareast-font-family:Arial'><span style='mso-list:Ignore'>&#8226;<span
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style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
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lang=EN-US>try Step 2 and 3</span></p>
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<p class=MsoNormal style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:l0 level1 lfo2;
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tab-stops:list 36.0pt'><![if !supportLists]><span lang=EN-US style='font-family:
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"Arial","sans-serif";mso-fareast-font-family:Arial'><span style='mso-list:Ignore'>&#8226;<span
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<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><u><span lang=EN-US>4.Genetic
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toggle switch to implement rewritable function <o:p></o:p></span></u></b></p>
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<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>
<p class=MsoNormal><span lang=EN-US><o:p>&nbsp;</o:p></span></p>

Revision as of 23:37, 21 October 2008

Home The Team The Project Parts Submitted to the Registry Modeling Notebook Japanese


Our project

　

We want to make a Coli Touch!!

　
　

We try to construct a bacterial ‘touch panel’ which is colored by the pressure.

 　
　

We name it Coli touch.

 　
　

Until iGEM2007, input style was only heat, small molecule and light. These input style have problems. For example, Heat input occur thermal gradient, small molecule input occur concentration gradient, light input depend on location of a container. So, we thought input style except these. That is “press”. Press input has an advantage that input signal travel uniformly in a container.

 　

Mechanism

　

We construct genetic circuits that can respond to pressure signal. Lac promoter was known as a pressure inducible promoter. Lac promoter is repressed by LacI protein. The repression is released by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside). Besides IPTG induction, pressure induction is possible. Under high pressure, the affinity of lac protein for lac operon decreases by transition of Lac protein from tetramer to dimer. Through this mechanism, Lac promoter is induced by pressure.

　
[サンプルの絵]

Strategy

　

We thought two projects, basic project and application project. Basic project consist of two categories. One is construction of pressure responsive genetic (1). The other is creating basic device for a touch display (2). Application project also consist of two categories. One is development of low pressure inducible promoter (3). The other is construction of pressure responsive toggle switch (4). In the future, we want to create the touch display made of E. coli.

　
[サンプルの絵]
　

We used these equipments (show figure) for pressure experiments.

　

1.Construction

　

We construct these plasmids (show figure).

　
　

Each part is constructed on low-copy plasmid “pSB6”.

　

2. Basic device

　

We created pressurizing device for first step of creating touch display. This device has two holes (show figure). Each hole contains tubes and water. Inside tubes E. coli is cultivated. Pressure can travel to inside tubes. One hole (B) is covered with a block made of an acrylic glass. (show figure) Therefore the hole is not pressurized by water. The other (A) is covered with a plastic tape (show figure). Therefore the hole is pressurized.

　

3. Development of low pressure inducible promoter

　

In the stage of basic project, very high pressure (30MPa) is required for pressure induction of lac promoter. But lower pressure response is needed for creating touch display. Therefore we develop lower pressure inducible promoter. We performed random mutagenesis to lac promoter and tried to screen promoters with FACS.

 　

4.Genetic toggle switch to implement rewritable function

 

 

 

 

 

 

 

・ genetic toggle switch

       - Writable function

Plac is induced under 30 MPa pressure

(PL is not induced by 30 MPa pressure)

Express GFP, CI, and CI represses PL

→ bright !!

- Erasable function

PL is induced by heat

Express LacI, Plac represses GFP

→ go out little by little…

 

Future work

      - Identify pressure model parameter from experimental data

      - Simulate pressure induction by pressure model and estimate

        promoter-RBS strength to implement rewritable function

 

 

 

 

 

 

 

 

 

 

 

 

Mathematical　model

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Retrieved from "http://2008.igem.org/Team:Tokyo_Tech"