http://2013.igem.org/wiki/index.php?title=Special:Contributions/Masashiohara&feed=atom&limit=50&target=Masashiohara&year=&month=2013.igem.org - User contributions [en]2024-03-28T19:40:41ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/ResultTeam:TMU-Tokyo/Notebook experiment/Result2013-09-28T04:22:46Z<p>Masashiohara: </p>
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<table class="LB" width="730" ><br />
<tr><td align="center">Fig.1</td><td align="center">Fig.2</td><td align="center">Fig.3</td></tr><br />
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<img class="work" src="https://static.igem.org/mediawiki/2013/9/9e/TMUresult-PCR0928.png"><br><br />
<p class="fig">Fig.4<p><br />
<br />
<br><br />
<h1> Assay1</h1><br />
<hr><br />
<br><br />
<p>We checked whether our device functioned definitely by the following assays. First, we measured activity of β-galactosidase using X-GAL (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-GAL is broken down by β-galactosidase and produces a pigment of the blue. When “Genomic Pythagorean Device” functions definitely, the expression of β-galactosidase should be induced by addition of arabinose and this reaction would not happen in the wild type of MG 1655. So, we first confirmed the expression of β-galactosidase after the addition of arabinose in both control group and experimental group. However, As a result, the expression of β-galactosidase wasn’t seen in experimental group. And colonies of control group are seen in light blue. We didn’t understand of this reason (Fig.1). <br />
<p>On the other hand, the addition of IPTG will induce the expression of β-galactosidase to both MG 1655 which have our device (experimental group or which haven’t it (control group). So, then we confirmed the expression of β-galactosidase after the addition of IPTG in both control group and experimental group. As a result, expression of β-galactosidase was seen in both groups (Fig.2). From this result, we could check that this reaction is reversible. Also, we could check that control group is not lacz - stock. (Control Experiment 1)<br />
<p>Then, we checked the expression of β‐galactosidase when we added nothing in LB medium (Fig.3). In this results, the expression of β-galactosidase wasn’t seen in both groups. (Control Experiment 2)<br />
<br />
<p>Therefore, we understand that some fragments in our device didn’t work as expected from this assay. In our device, plural chain reactions happen and finally β-galactosidase should be expressed. So, it was difficult to find where the cause that this device didn’t work was.</p><br />
<br><br />
<br />
<h1>Assay2</h1><br />
<hr><br />
<br><br />
<p>In order to pinpoint the problems in our device, we also carried out following experiments and checked whether inversion happens in Fragment1 by PCR (Fig.4). First, we designed primers 1, 2 and 3. And then we did PCR between primer 1 and 3, and primer 2 and 3. In order to examine to which direction pTet fronts, we checked the DNA band of the PCR product by electrophoresis. If the promoter inversion was happened by site specific recombination which mediated by HbiF, DNA band of PCR product which is performed between primer 1 and primer 3 only appear. DNA band of PCR product which is performed between primer 2 and primer3 should notbe appear.<br />
But the results don’t match to our prediction. The products of PCR which is performed either primer combinations didn't appear. The reason is not unclear so we will try to perform PCR with other new primer combinations.<br />
<p>Additionally, we performed PCR in order to check the deletion of lacI because of site specific recombination between two FRT sites in Fragment3. If lacI gene was deleted by the recombination, the PCR products which is performed between two outside primer of fragment3 become smaller than before deletion. If deletion didn’t happened, the PCR products has over 3kbm. On the other hand, the PCR products become only 1.4kb after deletion of lacI. <br />
<p>In our result, the size of all PCR products which are performed between two outside primer of fragment3 are over 3kb. So we suspect that our parts are not completely work. We should try other cultivating condition (for example cultivating over 8 hours), or specify which parts don’t work and fix it.<br />
<br />
<br />
<br />
</p><br />
<br><br />
<br><br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/ResultTeam:TMU-Tokyo/Notebook experiment/Result2013-09-28T04:18:51Z<p>Masashiohara: </p>
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<table class="LB" width="730" ><br />
<tr><td align="center">Fig.1</td><td align="center">Fig.2</td><td align="center">Fig.3</td></tr><br />
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<br><br />
<br><br />
<img class="work" src="https://static.igem.org/mediawiki/2013/9/9e/TMUresult-PCR0928.png"><br><br />
<p class="fig">Fig.4<p><br />
<br />
<br><br />
<h1> Assay1</h1><br />
<hr><br />
<p>We checked whether our device functioned definitely by the following assays. First, we measured activity of β-galactosidase using X-GAL (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-GAL is broken down by β-galactosidase and produces a pigment of the blue. When “Genomic Pythagorean Device” functions definitely, the expression of β-galactosidase should be induced by addition of arabinose and this reaction would not happen in the wild type of MG 1655. So, we first confirmed the expression of β-galactosidase after the addition of arabinose in both control group and experimental group. However, As a result, the expression of β-galactosidase wasn’t seen in experimental group. And colonies of control group are seen in light blue. We didn’t understand of this reason (Fig.1). <br />
<p>On the other hand, the addition of IPTG will induce the expression of β-galactosidase to both MG 1655 which have our device (experimental group or which haven’t it (control group). So, then we confirmed the expression of β-galactosidase after the addition of IPTG in both control group and experimental group. As a result, expression of β-galactosidase was seen in both groups (Fig.2). From this result, we could check that this reaction is reversible. Also, we could check that control group is not lacz - stock. (Control Experiment 1)<br />
<p>Then, we checked the expression of β‐galactosidase when we added nothing in LB medium (Fig.3). In this results, the expression of β-galactosidase wasn’t seen in both groups. (Control Experiment 2)<br />
<br />
<p>Therefore, we understand that some fragments in our device didn’t work as expected from this assay. In our device, plural chain reactions happen and finally β-galactosidase should be expressed. So, it was difficult to find where the cause that this device didn’t work was.</p><br />
<br><br />
<br />
<h1>Assay2</h1><br />
<hr><br />
<br />
<p>In order to pinpoint the problems in our device, we also carried out following experiments and checked whether inversion happens in Fragment1 by PCR (Fig.4). First, we designed primers 1, 2 and 3. And then we did PCR between primer 1 and 3, and primer 2 and 3. In order to examine to which direction pTet fronts, we checked the DNA band of the PCR product by electrophoresis. If the promoter inversion was happened by site specific recombination which mediated by HbiF, DNA band of PCR product which is performed between primer 1 and primer 3 only appear. DNA band of PCR product which is performed between primer 2 and primer3 should notbe appear.<br />
But the results don’t match to our prediction. The products of PCR which is performed either primer combinations didn't appear. The reason is not unclear so we will try to perform PCR with other new primer combinations.<br />
<p>Additionally, we performed PCR in order to check the deletion of lacI because of site specific recombination between two FRT sites in Fragment3. If lacI gene was deleted by the recombination, the PCR products which is performed between two outside primer of fragment3 become smaller than before deletion. If deletion didn’t happened, the PCR products has over 3kbm. On the other hand, the PCR products become only 1.4kb after deletion of lacI. <br />
<p>In our result, the size of all PCR products which are performed between two outside primer of fragment3 are over 3kb. So we suspect that our parts are not completely work. We should try other cultivating condition (for example cultivating over 8 hours), or specify which parts don’t work and fix it.<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/ResultTeam:TMU-Tokyo/Notebook experiment/Result2013-09-28T04:17:22Z<p>Masashiohara: </p>
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</div><br />
<table class="LB" width="730" ><br />
<tr><td align="center">Fig.1</td><td align="center">Fig.2</td><td align="center">Fig.3</td></tr><br />
</table><br />
<br><br />
<br><br />
<img class="work" src="https://static.igem.org/mediawiki/2013/9/9e/TMUresult-PCR0928.png"><br />
<br />
<br><br />
<h1> Assay1</h1><br />
<hr><br />
<p>We checked whether our device functioned definitely by the following assays. First, we measured activity of β-galactosidase using X-GAL (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-GAL is broken down by β-galactosidase and produces a pigment of the blue. When “Genomic Pythagorean Device” functions definitely, the expression of β-galactosidase should be induced by addition of arabinose and this reaction would not happen in the wild type of MG 1655. So, we first confirmed the expression of β-galactosidase after the addition of arabinose in both control group and experimental group. However, As a result, the expression of β-galactosidase wasn’t seen in experimental group. And colonies of control group are seen in light blue. We didn’t understand of this reason (Fig.1). <br />
<p>On the other hand, the addition of IPTG will induce the expression of β-galactosidase to both MG 1655 which have our device (experimental group or which haven’t it (control group). So, then we confirmed the expression of β-galactosidase after the addition of IPTG in both control group and experimental group. As a result, expression of β-galactosidase was seen in both groups (Fig.2). From this result, we could check that this reaction is reversible. Also, we could check that control group is not lacz - stock. (Control Experiment 1)<br />
<p>Then, we checked the expression of β‐galactosidase when we added nothing in LB medium (Fig.3). In this results, the expression of β-galactosidase wasn’t seen in both groups. (Control Experiment 2)<br />
<br />
<p>Therefore, we understand that some fragments in our device didn’t work as expected from this assay. In our device, plural chain reactions happen and finally β-galactosidase should be expressed. So, it was difficult to find where the cause that this device didn’t work was.</p><br />
<br><br />
<br />
<h1>Assay2</h1><br />
<hr><br />
<br />
<p>In order to pinpoint the problems in our device, we also carried out following experiments and checked whether inversion happens in Fragment1 by PCR (Fig.4). First, we designed primers 1, 2 and 3. And then we did PCR between primer 1 and 3, and primer 2 and 3. In order to examine to which direction pTet fronts, we checked the DNA band of the PCR product by electrophoresis. If the promoter inversion was happened by site specific recombination which mediated by HbiF, DNA band of PCR product which is performed between primer 1 and primer 3 only appear. DNA band of PCR product which is performed between primer 2 and primer3 should notbe appear.<br />
But the results don’t match to our prediction. The products of PCR which is performed either primer combinations didn't appear. The reason is not unclear so we will try to perform PCR with other new primer combinations.<br />
<p>Additionally, we performed PCR in order to check the deletion of lacI because of site specific recombination between two FRT sites in Fragment3. If lacI gene was deleted by the recombination, the PCR products which is performed between two outside primer of fragment3 become smaller than before deletion. If deletion didn’t happened, the PCR products has over 3kbm. On the other hand, the PCR products become only 1.4kb after deletion of lacI. <br />
<p>In our result, the size of all PCR products which are performed between two outside primer of fragment3 are over 3kb. So we suspect that our parts are not completely work. We should try other cultivating condition (for example cultivating over 8 hours), or specify which parts don’t work and fix it.<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/ResultTeam:TMU-Tokyo/Notebook experiment/Result2013-09-28T04:14:11Z<p>Masashiohara: </p>
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</div><br />
<br><br />
<br><br />
<img class="work" src="https://static.igem.org/mediawiki/2013/9/9e/TMUresult-PCR0928.png"><br />
<br />
<br><br />
<h1> Assay1</h1><br />
<hr><br />
<p>We checked whether our device functioned definitely by the following assays. First, we measured activity of β-galactosidase using X-GAL (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). X-GAL is broken down by β-galactosidase and produces a pigment of the blue. When “Genomic Pythagorean Device” functions definitely, the expression of β-galactosidase should be induced by addition of arabinose and this reaction would not happen in the wild type of MG 1655. So, we first confirmed the expression of β-galactosidase after the addition of arabinose in both control group and experimental group. However, As a result, the expression of β-galactosidase wasn’t seen in experimental group. And colonies of control group are seen in light blue. We didn’t understand of this reason (Fig.1). <br />
<p>On the other hand, the addition of IPTG will induce the expression of β-galactosidase to both MG 1655 which have our device (experimental group or which haven’t it (control group). So, then we confirmed the expression of β-galactosidase after the addition of IPTG in both control group and experimental group. As a result, expression of β-galactosidase was seen in both groups (Fig.2). From this result, we could check that this reaction is reversible. Also, we could check that control group is not lacz - stock. (Control Experiment 1)<br />
<p>Then, we checked the expression of β‐galactosidase when we added nothing in LB medium (Fig.3). In this results, the expression of β-galactosidase wasn’t seen in both groups. (Control Experiment 2)<br />
<br />
<p>Therefore, we understand that some fragments in our device didn’t work as expected from this assay. In our device, plural chain reactions happen and finally β-galactosidase should be expressed. So, it was difficult to find where the cause that this device didn’t work was.</p><br />
<br><br />
<br />
<h1>Assay2</h1><br />
<hr><br />
<br />
<p>In order to pinpoint the problems in our device, we also carried out following experiments and checked whether inversion happens in Fragment1 by PCR. First, we designed primers 1, 2 and 3. And then we did PCR between primer 1 and 3, and primer 2 and 3. In order to examine to which direction pTet fronts, we checked the DNA band of the PCR product by electrophoresis. If the promoter inversion was happened by site specific recombination which mediated by HbiF, DNA band of PCR product which is performed between primer 1 and primer 3 only appear. DNA band of PCR product which is performed between primer 2 and primer3 should notbe appear.<br />
But the results don’t match to our prediction. The products of PCR which is performed either primer combinations didn't appear. The reason is not unclear so we will try to perform PCR with other new primer combinations.<br />
<p>Additionally, we performed PCR in order to check the deletion of lacI because of site specific recombination between two FRT sites in Fragment3. (Fig.5) If lacI gene was deleted by the recombination, the PCR products which is performed between two outside primer of fragment3 become smaller than before deletion. If deletion didn’t happened, the PCR products has over 3kbm. On the other hand, the PCR products become only 1.4kb after deletion of lacI. <br />
<p>In our result, the size of all PCR products which are performed between two outside primer of fragment3 are over 3kb. So we suspect that our parts are not completely work. We should try other cultivating condition (for example cultivating over 8 hours), or specify which parts don’t work and fix it.<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/TeamTeam:TMU-Tokyo/Team2013-09-28T04:11:35Z<p>Masashiohara: </p>
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<Td style="padding: 20px;" Align="Center"><IMG Src="https://static.igem.org/mediawiki/igem.org/b/b8/TMUharuta.jpg" Width="120" Height="130"> </Td><br />
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<Td style="padding: 20px;" Align="Center"><IMG Src="https://static.igem.org/mediawiki/igem.org/a/a7/TMUokamoto.jpg"Width="120" Height="130"></Td><br />
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<Td style="padding: 20px;" Align="Center"><IMG Src="https://static.igem.org/mediawiki/igem.org/3/3d/TMUkato.jpg" Width="120" Height="130"></Td><br />
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<Td style="padding: 20px;" Align="Center"><IMG Src="https://static.igem.org/mediawiki/igem.org/4/41/TMUmurakami.jpg" Width="120" Height="130"></Td><br />
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<Td style="padding: 20px;" Align="Center"><IMG Src="https://static.igem.org/mediawiki/igem.org/7/71/TMUhukuda.jpg" Width="120" Height="130"></Td><br />
<br />
<br />
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<tr> <!--貢献度的に→Junichi Kato→Shin Haruta→Tkakashi Okamoto →Kimiko Hukuda→Noriak <br />
<br />
Murakami→Tsunaki Asano ?--> <br />
<td style="padding: 0px 10px 0px;" Align="center">Shin Haruta</td><br />
<td style="padding: 0px 10px 0px;" Align="center">Takashi Okamoto</td><br />
<td style="padding: 0px 10px 0px;" Align="center">Junichi Kato</td> <br />
<td style="padding: 0px 10px 0px;" Align="center">Tetsuaki Murakami</td><br />
<td style="padding: 0px 10px 0px;" Align="center">Kimiko Fukuda</td><br />
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</tr><br />
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<Br><Br><Br><br />
<br />
<p class="title"><b>Main Members</b></p><Br><br />
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<Table Border="0" Bordercolor="" Cellspacing="0" cellpadding="3"><br />
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<Td Align="Center"><IMG Src="https://static.igem.org/mediawiki/igem.org/7/72/TMUsakurai2.jpg" Width="185" Height="250"<br />
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<Td Align="Center"><IMG Src="https://static.igem.org/mediawiki/2013/6/64/TMUnakajima2.jpg"Width="185"height="250"<br />
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<Td Align="Center"><IMG Src="https://static.igem.org/mediawiki/igem.org/4/49/TMUtominaga.png" Width="185" Height="250"<br />
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<Td Align="Center"><IMG Src="https://static.igem.org/mediawiki/2013/f/fd/TMUOhara2.jpg" Width="185" Height="250" <br />
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<td Align="center">Eri Sakurai</td><br />
<td Align="center">Rena Nakajima</td><br />
<td Align="center">Kento Tominaga</td><br />
<td Align="center">Marina Ikeno</td><br />
<td Align="center">Masashi Ohara</td><br />
</tr> <br />
<tr id="post"><br />
<td class="post" font-size="small">Team Leader</td><br />
<td class="post">Accountant</td><br />
<td class="post">Project Leader</td><br />
<td class="post"></td><br />
<td class="post">Wiki writer</td><br />
<br />
</table><br />
</div><br />
<div class="grid_10"><br />
<p><Br><br />
</p><br />
</div><br />
<Br><br />
<Br><br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1 class="title" align="center"><b>Support Members</b></h1><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3b/Support_members.png" width="960" height="550"><br />
</div><br />
<div class="clear"></div><br />
<span style="line-height:50%"><Br></span><br />
<br />
<div class="container_12"><br />
<br />
<div class="grid_1"><br />
<p><Br></p><br />
</div><br />
<div class="grid_3"><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/62/TMUActsu.jpg"width="200" height="250"><br />
</div><br />
<div class="grid_4"><br />
<Br><p>←Takuya Akutsu<br><br />
(2012 TMU-Tokyo member)</p><Br><Br><Br><br />
<p align="right">→Yuki Komiya</p><br />
</div> <br />
<div class="grid_3"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/c/c9/TMUKomiya.jpg" width="200" height="250"><br />
</div><br />
<br />
<div class="grid_1"><br />
<p><Br></p><br />
</div><br />
<div class="clear"></div><br />
<br />
<Br><br />
<Br><br />
<br />
<br />
<Br><br />
<Br><br />
<Br><br />
<Br><br />
<Br><br />
<Br><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Template:Team:TMU-Tokyo/drop-menu.cssTemplate:Team:TMU-Tokyo/drop-menu.css2013-09-28T04:10:05Z<p>Masashiohara: </p>
<hr />
<div><html><br />
<style><br />
#dropmenu {<br />
list-style: none;<br />
align: left ;<br />
width: 1360px;<br />
height: 40px;<br />
margin: 30px <br />
padding: 0px;<br />
background: #7fffd4;<br />
<br />
<br />
border-radius: 3px 3px 0 0;<br />
}<br />
#dropmenu li {<br />
position: relative;<br />
width: 137px;<br />
float: left;<br />
margin: 0px;<br />
padding: 0;<br />
text-align: center;<br />
border-right: 1px solid #fff;<br />
<br />
}<br />
<br />
<br />
<br />
#dropmenu #leftbrock{<br />
width: 200px ;<br />
height: 40px;<br />
margin: 0px <br />
padding: 0px;<br />
border-right: 1px solid #fff;<br />
}<br />
#dropmenu #rightbrock{<br />
width: 200px ;<br />
height: 40px;<br />
margin: 0px <br />
padding: 0px;<br />
border-right: none ;} <br />
<br />
<br />
#dropmenu li a {<br />
display: block;<br />
margin: 0;<br />
padding: 15px 0 11px;<br />
color: #000;<br />
font-family: "calibri",sans-serif ;<br />
font-size: 18px;<br />
font-weight: bold ;<br />
line-height: 0.8;<br />
text-decoration: none;<br />
}<br />
#dropmenu li:hover > a{<br />
background: #dbfff6;<br />
color:#00000;<br />
}<br />
#dropmenu > li:hover > a{<br />
border-radius: 3px 3px 0 0;<br />
}<br />
#dropmenu li ul {<br />
position: absolute;<br />
top: 100%;<br />
left: 0;<br />
list-style: none;<br />
margin: 0;<br />
border-radius: 0 0 3px 3px;<br />
}<br />
<br />
<br />
#dropmenu li ul li{<br />
overflow: hidden;<br />
width: 150%;<br />
height: 0px;<br />
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-moz-transition: .2s;<br />
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-o-transition: .2s;<br />
-ms-transition: .2s;<br />
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#dropmenu li ul li a{<br />
padding: 13px 15px;<br />
background: #dbfff6;<br />
text-align: center;<br />
font-size: 10px;<br />
font-weight: normal;<br />
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}<br />
<br />
<br />
<br />
#dropmenu li:hover ul li{<br />
overflow: visible;<br />
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border-top: 0px solid #dbfff6;<br />
border-bottom: 0px solid #dbfff6;<br />
}<br />
<br />
#dropmenu li:hover ul li:hover a{<br />
background: #7fffd4;<br />
color:#00000<br />
}<br />
<br />
#dropmenu li:hover ul li:first-child{<br />
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#dropmenu li:hover ul li:last-child{<br />
border-bottom: 0;<br />
}<br />
#dropmenu li:hover ul li:last-child a{<br />
border-radius: 0 0 3px 3px;<br />
}<br />
<br />
</style><br />
</html><br />
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<ul><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Human_contribution">Collaboration</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Human_spread">Promote Synthetic Biology</a></li><br />
<br />
</ul><br />
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<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety">Safety</a><br />
<ul><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_Safety">Lab safety</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety/Questions">Questions</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Achievement">Achievement</a><br />
<ul><br />
</ul><br />
</li><br />
<li class="note"><a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_menu">Notebook</a><br />
<ul><br />
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<br />
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</ul><br />
</div><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Template:Team:TMU-Tokyo/drop-menu.cssTemplate:Team:TMU-Tokyo/drop-menu.css2013-09-28T04:09:51Z<p>Masashiohara: </p>
<hr />
<div><html><br />
<style><br />
#dropmenu {<br />
list-style: none;<br />
align: left ;<br />
width: 1360px;<br />
height: 40px;<br />
margin: 30px <br />
padding: 0px;<br />
background: #7fffd4;<br />
<br />
<br />
border-radius: 3px 3px 0 0;<br />
}<br />
#dropmenu li {<br />
position: relative;<br />
width: 137px;<br />
float: left;<br />
margin: 0px;<br />
padding: 0;<br />
text-align: center;<br />
border-right: 1px solid #fff;<br />
<br />
}<br />
<br />
<br />
<br />
#dropmenu #leftbrock{<br />
width: 200px ;<br />
height: 40px;<br />
margin: 0px <br />
padding: 0px;<br />
border-right: 1px solid #fff;<br />
}<br />
#dropmenu #rightbrock{<br />
width: 200px ;<br />
height: 40px;<br />
margin: 0px <br />
padding: 0px;<br />
border-right: none ;} <br />
<br />
<br />
#dropmenu li a {<br />
display: block;<br />
margin: 0;<br />
padding: 15px 0 11px;<br />
color: #000;<br />
font-family: "calibri",sans-serif ;<br />
font-size: 18px;<br />
font-weight: bold ;<br />
line-height: 0.8;<br />
text-decoration: none;<br />
}<br />
#dropmenu li:hover > a{<br />
background: #dbfff6;<br />
color:#00000;<br />
}<br />
#dropmenu > li:hover > a{<br />
border-radius: 3px 3px 0 0;<br />
}<br />
#dropmenu li ul {<br />
position: absolute;<br />
top: 100%;<br />
left: 0;<br />
list-style: none;<br />
margin: 0;<br />
border-radius: 0 0 3px 3px;<br />
}<br />
<br />
<br />
#dropmenu li ul li{<br />
overflow: hidden;<br />
width: 150%;<br />
height: 0px;<br />
color: #000000;<br />
-moz-transition: .2s;<br />
-webkit-transition: .2s;<br />
-o-transition: .2s;<br />
-ms-transition: .2s;<br />
transition: .2s;<br />
}<br />
#dropmenu li ul li a{<br />
padding: 13px 15px;<br />
background: #dbfff6;<br />
text-align: center;<br />
font-size: 10px;<br />
font-weight: normal;<br />
<br />
}<br />
<br />
<br />
<br />
#dropmenu li:hover ul li{<br />
overflow: visible;<br />
height: 40px;<br />
border-top: 0px solid #dbfff6;<br />
border-bottom: 0px solid #dbfff6;<br />
}<br />
<br />
#dropmenu li:hover ul li:hover a{<br />
background: #7fffd4;<br />
color:#00000<br />
}<br />
<br />
#dropmenu li:hover ul li:first-child{<br />
border-top: 0;<br />
}<br />
#dropmenu li:hover ul li:last-child{<br />
border-bottom: 0;<br />
}<br />
#dropmenu li:hover ul li:last-child a{<br />
border-radius: 0 0 3px 3px;<br />
}<br />
<br />
</style><br />
</html><br />
<br />
<html><br />
<div id="content"><br />
<ul id="dropmenu"><br />
<li id="leftbrock"><br />
<p><Br></p><br />
</li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo">Home</a><br />
<br />
</li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Team_Team">Team</a><br />
<ul><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Team">Members</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Team_attribution">Attribution</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Project">Project</a><br />
<ul><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Idea">Idea</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/NEWS">Breakthrough</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device">Pythagorean Device</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Parts">Parts</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Future_Plan">Future Plan</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Reference">Reference</a></li><br />
</ul><br />
</li><br />
<li class="Human"><a href="https://2013.igem.org/Team:TMU-Tokyo/HumanPractice">HumanPractice</a><br />
<ul><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Human_contribution">Collaboration</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Human_spread">Promote Synthetic Biology</a></li><br />
<br />
</ul><br />
</li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety">Safety</a><br />
<ul><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_Safety">Lab safety</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety/Questions">Questions</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Achievement">Achievement</a><br />
<ul><br />
</ul><br />
</li><br />
<li class="note"><a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_menu">Notebook</a><br />
<ul><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook">Protocol</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment">Experiment</a></li><br />
<li><a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/Result">Result</a></li><br />
</ul><br />
</li><br />
<li id="rightbrock"> <br />
<p><Br</p><br />
</li><br />
<br />
<br />
<br />
</ul><br />
</div><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_DeviceTeam:TMU-Tokyo/Project/Pythagorean Device2013-09-28T04:07:41Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/Home.css}}<br />
{{Template:Team:TMU-Tokyo/link}}<br />
<br />
<html><br />
<head><style><br />
img.work {display:block;<br />
margin-left:auto;<br />
margin-right:auto;<br />
}<br />
#RED li {list-style-type:decimal;<br />
margin-left:80px;<br />
font-size:medium;<br />
}<br />
</style></head><br />
<br />
<br><br />
<br><br />
<br />
<div class="container_12"><br />
<h1>Overview</h1><br />
<hr><br />
<br><br />
<p>According to “Breakthrough”, we inserted “Genomic Pythagorean Device”, which we designed in a genome of <i>E.coli</i>. </p><br />
<p>“Pythagorean Devices” are very familiar to many Japanese people because Japanese famous educational TV program named “Pythagorean Switch” introduces a lot kind of them. They are known as "Rube Goldberg machines" in the US. Pythagorean Devices are deliberately over-engineered or overdone systems that perform very simple task in very complicated process and they usually include some chain reactions. We can enjoy this complicated process. In this year, we tried to design and make the device which is like “Pythagorean Device” in a genome of <i>E.coli</i>. We are very interested in site specific recombination systems in bacteria, so we used lots kind of site specific recombination enzymes in our device. They were useful to make the process of this device more complicated.</p><br />
<p> Therefore, we succeeded in making a complicated chain reaction course in a genome of <i>E.coli</i>.</p><br />
<p>Also we experimentally checked whether this device works or not.</p><br />
<br><br />
<p style="margin-left:250px;">↓What is “Pythagorean Device”? –Like this!</p><br />
<br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_3"><br />
<p><br></p><br />
</div><br />
<div class="grid_5"><br />
<iframe width="420" height="315" src="//www.youtube.com/embed/bg0BR4FGHQc?rel=0" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</div><br />
<div class="grid_4"><br />
<p><br></p><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br><br />
<br><br />
<div class="container_12"><br />
<h1>How dose it work?</h1><br />
<hr><br />
<br><br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. </p><br />
<br><br />
<img class="work" width="520" height="385" src="https://static.igem.org/mediawiki/2013/1/17/TMUFig.1.png"><br><br />
<p> We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.</p><br />
<br><br />
<img class="work" width="640" height="222"src="https://static.igem.org/mediawiki/igem.org/d/d8/TMU-PDfig2.png"><br />
<br><br />
<img class="work" width="640" height="198"src="https://static.igem.org/mediawiki/igem.org/6/67/TMU-PDfig3.png"><br />
<br><br />
<br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. <br />
We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.<br />
<br />
We inserted them in a genome by lambda bacteriophage recombination system, “RED”.<br />
Then, how does this device work?<br />
</p><br />
<br><br />
<ol id="RED"><br />
<li> A large quantity of lacI is expressed by lacI gene.</li><br />
<li>LacI represses plac.</li><br />
<li>By adding arabinose, pBad is activated and site specific recombination enzyme, HbiF comes to be expressed.<br />
<li>Because of HbiF, inversion happens between the site of IRL and IRR. And then site specific recombination enzyme named Flp comes to be expressed.</li><br />
<li>Site specific recombination happens and the sequence between the 2 site of FRT is left off by Flp.</li><br />
<li>Repression of plac becomes off by deleting of lacIq gene and then β-gal comes to be expressed.</li><br />
<li>Surrounded <i>E.coli</i> in the form of Japanese character of “ピ” on the plate which x-gal was added in beforehand.</li><br />
<li>TThe form of the character of “ピ” comes to blue.</li><br />
</ol><br />
<br><br />
<br />
<div class="clear"></div> <br />
<br />
<br> <br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<br />
<br />
<img class="work" width="850" height="1328"src="https://static.igem.org/mediawiki/2013/d/d7/TMUPD_Flow.2.png"><br />
<br />
<br><br />
<br><br />
<br />
<h1>Result</h1><br />
<hr><br />
<br><br />
<a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/Result" style="font-size:38pt;margin-lefrt:600px;">⇒Result</a><br />
<br><br><br><br />
</div><br />
<br />
<!---<br />
<div class="container_12"><br />
<h1></h1><br />
<hr>--><br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_DeviceTeam:TMU-Tokyo/Project/Pythagorean Device2013-09-28T04:06:46Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/Home.css}}<br />
{{Template:Team:TMU-Tokyo/link}}<br />
<br />
<html><br />
<head><style><br />
img.work {display:block;<br />
margin-left:auto;<br />
margin-right:auto;<br />
}<br />
#RED li {list-style-type:decimal;<br />
margin-left:80px;<br />
font-size:medium;<br />
}<br />
</style></head><br />
<br />
<br><br />
<br><br />
<br />
<div class="container_12"><br />
<h1>Overview</h1><br />
<hr><br />
<br><br />
<p>According to “Breakthrough”, we inserted “Genomic Pythagorean Device”, which we designed in a genome of <i>E.coli</i>. </p><br />
<p>“Pythagorean Devices” are very familiar to many Japanese people because Japanese famous educational TV program named “Pythagorean Switch” introduces a lot kind of them. They are known as "Rube Goldberg machines" in the US. Pythagorean Devices are deliberately over-engineered or overdone systems that perform very simple task in very complicated process and they usually include some chain reactions. We can enjoy this complicated process. In this year, we tried to design and make the device which is like “Pythagorean Device” in a genome of <i>E.coli</i>. We are very interested in site specific recombination systems in bacteria, so we used lots kind of site specific recombination enzymes in our device. They were useful to make the process of this device more complicated.</p><br />
<p> Therefore, we succeeded in making a complicated chain reaction course in a genome of <i>E.coli</i>.</p><br />
<p>Also we experimentally checked whether this device works or not.</p><br />
<br><br />
<p style="margin-left:250px;">↓What is “Pythagorean Device”? –Like this!</p><br />
<br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_3"><br />
<p><br></p><br />
</div><br />
<div class="grid_5"><br />
<iframe width="420" height="315" src="//www.youtube.com/embed/bg0BR4FGHQc?rel=0" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</div><br />
<div class="grid_4"><br />
<p><br></p><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br><br />
<br><br />
<div class="container_12"><br />
<h1>How dose it work?</h1><br />
<hr><br />
<br><br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. </p><br />
<br><br />
<img class="work" width="520" height="385" src="https://static.igem.org/mediawiki/2013/1/17/TMUFig.1.png"><br><br />
<p> We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.</p><br />
<br><br />
<img class="work" width="640" height="222"src="https://static.igem.org/mediawiki/igem.org/d/d8/TMU-PDfig2.png"><br />
<br><br />
<img class="work" width="640" height="198"src="https://static.igem.org/mediawiki/igem.org/6/67/TMU-PDfig3.png"><br />
<br><br />
<br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. <br />
We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.<br />
<br />
We inserted them in a genome by lambda bacteriophage recombination system, “RED”.<br />
Then, how does this device work?<br />
</p><br />
<br><br />
<ol id="RED"><br />
<li> A large quantity of lacI is expressed by lacI gene.</li><br />
<li>LacI represses plac.</li><br />
<li>By adding arabinose, pBad is activated and site specific recombination enzyme, HbiF comes to be expressed.<br />
<li>Because of HbiF, inversion happens between the site of IRL and IRR. And then site specific recombination enzyme named Flp comes to be expressed.</li><br />
<li>Site specific recombination happens and the sequence between the 2 site of FRT is left off by Flp.</li><br />
<li>Repression of plac becomes off by deleting of lacIq gene and then β-gal comes to be expressed.</li><br />
<li>Surrounded <i>E.coli</i> in the form of Japanese character of “ピ” on the plate which x-gal was added in beforehand.</li><br />
<li>TThe form of the character of “ピ” comes to blue.</li><br />
</ol><br />
<br><br />
<br />
<div class="clear"></div> <br />
<br />
<br> <br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<br />
<br />
<img class="work" width="850" height="1328"src="https://static.igem.org/mediawiki/2013/d/d7/TMUPD_Flow.2.png"><br />
<br />
<br><br />
<br><br />
<br />
<h1>Result</h1><br />
<hr><br />
<br><br />
<a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/Result" style="font-size:38pt;">Result</a><br />
<br><br><br><br />
</div><br />
<br />
<!---<br />
<div class="container_12"><br />
<h1></h1><br />
<hr>--><br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_DeviceTeam:TMU-Tokyo/Project/Pythagorean Device2013-09-28T04:06:15Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/Home.css}}<br />
{{Template:Team:TMU-Tokyo/link}}<br />
<br />
<html><br />
<head><style><br />
img.work {display:block;<br />
margin-left:auto;<br />
margin-right:auto;<br />
}<br />
#RED li {list-style-type:decimal;<br />
margin-left:80px;<br />
font-size:medium;<br />
}<br />
</style></head><br />
<br />
<br><br />
<br><br />
<br />
<div class="container_12"><br />
<h1>Overview</h1><br />
<hr><br />
<br><br />
<p>According to “Breakthrough”, we inserted “Genomic Pythagorean Device”, which we designed in a genome of <i>E.coli</i>. </p><br />
<p>“Pythagorean Devices” are very familiar to many Japanese people because Japanese famous educational TV program named “Pythagorean Switch” introduces a lot kind of them. They are known as "Rube Goldberg machines" in the US. Pythagorean Devices are deliberately over-engineered or overdone systems that perform very simple task in very complicated process and they usually include some chain reactions. We can enjoy this complicated process. In this year, we tried to design and make the device which is like “Pythagorean Device” in a genome of <i>E.coli</i>. We are very interested in site specific recombination systems in bacteria, so we used lots kind of site specific recombination enzymes in our device. They were useful to make the process of this device more complicated.</p><br />
<p> Therefore, we succeeded in making a complicated chain reaction course in a genome of <i>E.coli</i>.</p><br />
<p>Also we experimentally checked whether this device works or not.</p><br />
<br><br />
<p style="margin-left:250px;">↓What is “Pythagorean Device”? –Like this!</p><br />
<br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_3"><br />
<p><br></p><br />
</div><br />
<div class="grid_5"><br />
<iframe width="420" height="315" src="//www.youtube.com/embed/bg0BR4FGHQc?rel=0" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</div><br />
<div class="grid_4"><br />
<p><br></p><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br><br />
<br><br />
<div class="container_12"><br />
<h1>How dose it work?</h1><br />
<hr><br />
<br><br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. </p><br />
<br><br />
<img class="work" width="520" height="385" src="https://static.igem.org/mediawiki/2013/1/17/TMUFig.1.png"><br><br />
<p> We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.</p><br />
<br><br />
<img class="work" width="640" height="222"src="https://static.igem.org/mediawiki/igem.org/d/d8/TMU-PDfig2.png"><br />
<br><br />
<img class="work" width="640" height="198"src="https://static.igem.org/mediawiki/igem.org/6/67/TMU-PDfig3.png"><br />
<br><br />
<br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. <br />
We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.<br />
<br />
We inserted them in a genome by lambda bacteriophage recombination system, “RED”.<br />
Then, how does this device work?<br />
</p><br />
<br><br />
<ol id="RED"><br />
<li> A large quantity of lacI is expressed by lacI gene.</li><br />
<li>LacI represses plac.</li><br />
<li>By adding arabinose, pBad is activated and site specific recombination enzyme, HbiF comes to be expressed.<br />
<li>Because of HbiF, inversion happens between the site of IRL and IRR. And then site specific recombination enzyme named Flp comes to be expressed.</li><br />
<li>Site specific recombination happens and the sequence between the 2 site of FRT is left off by Flp.</li><br />
<li>Repression of plac becomes off by deleting of lacIq gene and then β-gal comes to be expressed.</li><br />
<li>Surrounded <i>E.coli</i> in the form of Japanese character of “ピ” on the plate which x-gal was added in beforehand.</li><br />
<li>TThe form of the character of “ピ” comes to blue.</li><br />
</ol><br />
<br><br />
<br />
<div class="clear"></div> <br />
<br />
<br> <br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<br />
<br />
<img class="work" width="850" height="1328"src="https://static.igem.org/mediawiki/2013/d/d7/TMUPD_Flow.2.png"><br />
<br />
<br><br />
<br><br />
<br />
<h1>Result</h1><br />
<hr><br />
<a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/Result" style="font-size:38pt;">Result</a><br />
<br />
</div><br />
<br />
<!---<br />
<div class="container_12"><br />
<h1></h1><br />
<hr>--><br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_DeviceTeam:TMU-Tokyo/Project/Pythagorean Device2013-09-28T04:04:28Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/Home.css}}<br />
<br />
<html><br />
<head><style><br />
img.work {display:block;<br />
margin-left:auto;<br />
margin-right:auto;<br />
}<br />
#RED li {list-style-type:decimal;<br />
margin-left:80px;<br />
font-size:medium;<br />
}<br />
</style></head><br />
<br />
<br><br />
<br><br />
<br />
<div class="container_12"><br />
<h1>Overview</h1><br />
<hr><br />
<br><br />
<p>According to “Breakthrough”, we inserted “Genomic Pythagorean Device”, which we designed in a genome of <i>E.coli</i>. </p><br />
<p>“Pythagorean Devices” are very familiar to many Japanese people because Japanese famous educational TV program named “Pythagorean Switch” introduces a lot kind of them. They are known as "Rube Goldberg machines" in the US. Pythagorean Devices are deliberately over-engineered or overdone systems that perform very simple task in very complicated process and they usually include some chain reactions. We can enjoy this complicated process. In this year, we tried to design and make the device which is like “Pythagorean Device” in a genome of <i>E.coli</i>. We are very interested in site specific recombination systems in bacteria, so we used lots kind of site specific recombination enzymes in our device. They were useful to make the process of this device more complicated.</p><br />
<p> Therefore, we succeeded in making a complicated chain reaction course in a genome of <i>E.coli</i>.</p><br />
<p>Also we experimentally checked whether this device works or not.</p><br />
<br><br />
<p style="margin-left:250px;">↓What is “Pythagorean Device”? –Like this!</p><br />
<br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_3"><br />
<p><br></p><br />
</div><br />
<div class="grid_5"><br />
<iframe width="420" height="315" src="//www.youtube.com/embed/bg0BR4FGHQc?rel=0" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</div><br />
<div class="grid_4"><br />
<p><br></p><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br><br />
<br><br />
<div class="container_12"><br />
<h1>How dose it work?</h1><br />
<hr><br />
<br><br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. </p><br />
<br><br />
<img class="work" width="520" height="385" src="https://static.igem.org/mediawiki/2013/1/17/TMUFig.1.png"><br><br />
<p> We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.</p><br />
<br><br />
<img class="work" width="640" height="222"src="https://static.igem.org/mediawiki/igem.org/d/d8/TMU-PDfig2.png"><br />
<br><br />
<img class="work" width="640" height="198"src="https://static.igem.org/mediawiki/igem.org/6/67/TMU-PDfig3.png"><br />
<br><br />
<br />
<p>“Genomic Pythagorean Device” is constructed by following 4 fragments. <br />
We made these fragments by over rap extension PCR and inserted in a genome of <i>E.coli</i> according to the following figures.<br />
<br />
We inserted them in a genome by lambda bacteriophage recombination system, “RED”.<br />
Then, how does this device work?<br />
</p><br />
<br><br />
<ol id="RED"><br />
<li> A large quantity of lacI is expressed by lacI gene.</li><br />
<li>LacI represses plac.</li><br />
<li>By adding arabinose, pBad is activated and site specific recombination enzyme, HbiF comes to be expressed.<br />
<li>Because of HbiF, inversion happens between the site of IRL and IRR. And then site specific recombination enzyme named Flp comes to be expressed.</li><br />
<li>Site specific recombination happens and the sequence between the 2 site of FRT is left off by Flp.</li><br />
<li>Repression of plac becomes off by deleting of lacIq gene and then β-gal comes to be expressed.</li><br />
<li>Surrounded <i>E.coli</i> in the form of Japanese character of “ピ” on the plate which x-gal was added in beforehand.</li><br />
<li>TThe form of the character of “ピ” comes to blue.</li><br />
</ol><br />
<br><br />
<br />
<div class="clear"></div> <br />
<br />
<br> <br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<br />
<br />
<img class="work" width="850" height="1328"src="https://static.igem.org/mediawiki/2013/d/d7/TMUPD_Flow.2.png"><br />
<br />
<br><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/Result" style="font-size:18pt;">Result</a><br />
<br />
</div><br />
<br />
<!---<br />
<div class="container_12"><br />
<h1></h1><br />
<hr>--><br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T03:58:34Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/Human_contribution.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<br />
<br />
<br />
<html><br />
<Br><Br><br />
<div class="container_12"><br />
<h1>Safty Check List</h1><br />
<hr><br />
<br />
<br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_5"><br />
<br><br />
<br><br />
<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
<br />
</p><br />
</div><br />
<br />
<div class="grid_5"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4f/TMUSafty_checklist.jpg" width="360" height="400" > <br />
<br />
<br />
</div><br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<div class="clear"></div><br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Safety Guidance by our instructors</h1><br />
<hr><br />
<br><br />
<table id=lab><br />
<tr><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Plant Photoregulation Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<div class="grid_12"><br />
<br><br />
<br><br />
<p><br><br />
<p>We thought that it was very important to experiment safely and wrote this policy on“iGEM 2013 basic safety form”too. Our team members think that the experiments done in dangerous way are meaningless. Therefore, we took care to protect biodiversity against genetically modified organisms (GMOs). Also we really took care our project didn't have any bad effect on environment and human body.<br><br />
<p>Therefore, we received the safety guidsnce from our instructors to experiment safely at laboratories. In the guidance, we are taught that how we should write the experimental data in notebooks and how we should use experimental devices or chemicals are in the laboratories.<br><br />
Also we’ve experimented along their lab’s rules.<br><br />
<br />
<br />
<br><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T03:53:56Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/Human_contribution.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<br />
<br />
<br />
<html><br />
<Br><Br><br />
<div class="container_12"><br />
<h1>Safty Check List</h1><br />
<hr><br />
<br />
<br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_5"><br />
<br><br />
<br><br />
<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
<br />
</p><br />
</div><br />
<br />
<div class="grid_5"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4f/TMUSafty_checklist.jpg" width="360" height="400" > <br />
<br />
<br />
</div><br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<div class="clear"></div><br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Safety Guidance by our instructors</h1><br />
<hr><br />
<br><br />
<table id=lab><br />
<tr><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Plant Photoregulation Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<div class="grid_12"><br />
<br><br />
<br><br />
<p><br><br />
<p>We thought that to experiment safely is very important like we wrote “iGEM 2013 basic safety form.” Not safe experiments are meaningless. We must protect biodiversity against genetically modified organisms (GMOs). Also we must not impact badly on our body.<br><br />
<p>Therefore, we received the safety guidsnce from our instructors to experiment safely at laboratories. In the guidance, we are taught that how we should write the experimental data in notebooks and how we should use experimental devices or chemicals are in the laboratories.<br><br />
Also we’ve experimented along their lab’s rules.<br><br />
<br />
<br />
<br><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T03:52:32Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/Human_contribution.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<br />
<style><br />
#lab{display:block;<br />
margin-left:80px;<br />
margin-right:auto;}<br />
#lab td {border:1px;<br />
font-size:medium;<br />
padding:0 5px;}<br />
</style><br />
<br />
<html><br />
<Br><Br><br />
<div class="container_12"><br />
<h1>Safty Check List</h1><br />
<hr><br />
<br />
<br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_5"><br />
<br><br />
<br><br />
<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
<br />
</p><br />
</div><br />
<br />
<div class="grid_5"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4f/TMUSafty_checklist.jpg" width="360" height="400" > <br />
<br />
<br />
</div><br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<div class="clear"></div><br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Safety Guidance by our instructors</h1><br />
<hr><br />
<br><br />
<table id=lab><br />
<tr><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Plant Photoregulation Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<div class="grid_12"><br />
<br><br />
<br><br />
<p><br><br />
<p>We thought that to experiment safely is very important like we wrote “iGEM 2013 basic safety form.” Not safe experiments are meaningless. We must protect biodiversity against genetically modified organisms (GMOs). Also we must not impact badly on our body.<br><br />
<p>Therefore, we received the safety guidsnce from our instructors to experiment safely at laboratories. In the guidance, we are taught that how we should write the experimental data in notebooks and how we should use experimental devices or chemicals are in the laboratories.<br><br />
Also we’ve experimented along their lab’s rules.<br><br />
<br />
<br />
<br><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T03:49:48Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/Human_contribution.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<br />
<br />
<br />
<html><br />
<style><br />
#lab{display:block;<br />
margin-left:80px;<br />
margin-right:auto;}<br />
#lab td {border:1px;<br />
font-size:medium;<br />
padding:0 5px;}<br />
</style><br />
<br />
<Br><Br><br />
<div class="container_12"><br />
<h1>Safty Check List</h1><br />
<hr><br />
<br />
<br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_5"><br />
<br><br />
<br><br />
<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
<br />
</p><br />
</div><br />
<br />
<div class="grid_5"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4f/TMUSafty_checklist.jpg" width="360" height="400" > <br />
<br />
<br />
</div><br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<div class="clear"></div><br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Safety Guidance by our instructors</h1><br />
<hr><br />
<br><br />
<table id="lab"><br />
<tr><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Plant Photoregulation Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<div class="grid_12"><br />
<br><br />
<br><br />
<p><br><br />
<p>We thought that to experiment safely is very important like we wrote “iGEM 2013 basic safety form.” Not safe experiments are meaningless. We must protect biodiversity against genetically modified organisms (GMOs). Also we must not impact badly on our body.<br><br />
<p>Therefore, we received the safety guidsnce from our instructors to experiment safely at laboratories. In the guidance, we are taught that how we should write the experimental data in notebooks and how we should use experimental devices or chemicals are in the laboratories.<br><br />
Also we’ve experimented along their lab’s rules.<br><br />
<br />
<br />
<br><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T03:47:27Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/Human_contribution.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<br />
<br />
<br />
<html><br />
<style><br />
#lab{display:block;<br />
margin-left:80px;<br />
margin-right:auto;}<br />
#lab td {font-size:medium;}<br />
</style><br />
<br />
<Br><Br><br />
<div class="container_12"><br />
<h1>Safty Check List</h1><br />
<hr><br />
<br />
<br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_5"><br />
<br><br />
<br><br />
<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
<br />
</p><br />
</div><br />
<br />
<div class="grid_5"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4f/TMUSafty_checklist.jpg" width="360" height="400" > <br />
<br />
<br />
</div><br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<div class="clear"></div><br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Safety Guidance by our instructors</h1><br />
<hr><br />
<br><br />
<table id="lab"><br />
<tr cellpadding="3px"><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Plant Photoregulation Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<div class="grid_12"><br />
<br><br />
<br><br />
<p><br><br />
<p>We thought that to experiment safely is very important like we wrote “iGEM 2013 basic safety form.” Not safe experiments are meaningless. We must protect biodiversity against genetically modified organisms (GMOs). Also we must not impact badly on our body.<br><br />
<p>Therefore, we received the safety guidsnce from our instructors to experiment safely at laboratories. In the guidance, we are taught that how we should write the experimental data in notebooks and how we should use experimental devices or chemicals are in the laboratories.<br><br />
Also we’ve experimented along their lab’s rules.<br><br />
<br />
<br />
<br><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T03:45:33Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/Human_contribution.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<br />
<br />
<br />
<html><br />
<style><br />
#lab{display:block;<br />
margin-left:100px;<br />
margin-right:auto;}<br />
#lab td {font-size:medium;}<br />
</style><br />
<br />
<Br><Br><br />
<div class="container_12"><br />
<h1>Safty Check List</h1><br />
<hr><br />
<br />
<br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_5"><br />
<br><br />
<br><br />
<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
<br />
</p><br />
</div><br />
<br />
<div class="grid_5"><br />
<img src="https://static.igem.org/mediawiki/2013/4/4f/TMUSafty_checklist.jpg" width="360" height="400" > <br />
<br />
<br />
</div><br />
<div class="grid_1"><br />
<p><br></p><br />
</div><br />
<div class="clear"></div><br />
<br />
<br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Safety Guidance by our instructors</h1><br />
<hr><br />
<br><br />
<table id="lab"><br />
<tr><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Plant Photoregulation Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<div class="grid_12"><br />
<br><br />
<br><br />
<p><br><br />
<p>We thought that to experiment safely is very important like we wrote “iGEM 2013 basic safety form.” Not safe experiments are meaningless. We must protect biodiversity against genetically modified organisms (GMOs). Also we must not impact badly on our body.<br><br />
<p>Therefore, we received the safety guidsnce from our instructors to experiment safely at laboratories. In the guidance, we are taught that how we should write the experimental data in notebooks and how we should use experimental devices or chemicals are in the laboratories.<br><br />
Also we’ve experimented along their lab’s rules.<br><br />
<br />
<br />
<br><br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/ProjectTeam:TMU-Tokyo/Project2013-09-28T03:37:15Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/link}}<br />
<br />
<html><br />
<br><br />
<br />
<div class="container_12"><br />
<img width="960" height="300" src="https://static.igem.org/mediawiki/igem.org/e/ef/TMUDoor_project.png"><br />
<br><br />
</div><br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_2"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_10"><br />
<br><br />
<ul><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Idea">Idea</a></p></li><br />
<p>Our fundamental idea and background are written here.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/NEWS">Breakthrough</a></li></p><br />
<p>We put parts not in plasmid but it in a genome of E.coli.</p><p>This page explain the method we named "Breakthrough".</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device">Pythagorean_Device</a></li></p><br />
<p>If you read this page, you will understand what the Pythagorean_Device is. </p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Parts">Parts</a></li></p><br />
<p>You find what part we submitted.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Future_Plan">Future plan</a></li></p><br />
<p>You can see the design of a more complicated device</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Reference">Reference</a></li></p><br />
<p></p><br><br />
</ul><br />
<br />
</div></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/ProjectTeam:TMU-Tokyo/Project2013-09-28T03:32:59Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/link}}<br />
<br />
<html><br />
<br><br />
<br />
<div class="container_12"><br />
<img width="960" height="300" src="https://static.igem.org/mediawiki/igem.org/e/ef/TMUDoor_project.png"><br />
<br><br />
</div><br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_2"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_10"><br />
<br><br />
<ul><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Idea">Idea</a></p></li><br />
<p>Our fundamental idea and background are written here.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/NEWS">Breakthrough</a></li></p><br />
<p>We put parts not in plasmid but it in a genome of E.coli. This page explain the method we named "Breakthrough".</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device">Pythagorean_Device</a></li></p><br />
<p>If you read this page, you will understand what the Pythagorean_Device is. </p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Parts">Parts</a></li></p><br />
<p>You find what part we submitted.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Future_Plan">Future plan</a></li></p><br />
<p>You can see the design of a more complicated device</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Reference">Reference</a></li></p><br />
<p></p><br><br />
</ul><br />
<br />
</div></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/ProjectTeam:TMU-Tokyo/Project2013-09-28T03:27:01Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/link}}<br />
<br />
<html><br />
<br><br />
<br />
<div class="container_12"><br />
<img width="960" height="300" src="https://static.igem.org/mediawiki/igem.org/e/ef/TMUDoor_project.png"><br />
<br><br />
</div><br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_2"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_10"><br />
<br><br />
<ul><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Idea">Idea</a></p></li><br />
<p>Our fundamental idea and background are written here.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/NEWS">Breakthrough</a></li></p><br />
<p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device">Pythagorean_Device</a></li></p><br />
<p>If you read this page, you will understand what the Pythagorean_Device is. </p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Parts">Parts</a></li></p><br />
<p>You find what part we submitted.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Future_Plan">Future plan</a></li></p><br />
<p>You can see the design of a more complicated device</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Reference">Reference</a></li></p><br />
<p></p><br><br />
</ul><br />
<br />
</div></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/ProjectTeam:TMU-Tokyo/Project2013-09-28T03:23:54Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
{{Template:Team:TMU-Tokyo/link}}<br />
<br />
<html><br />
<br><br />
<br />
<div class="container_12"><br />
<img width="960" height="300" src="https://static.igem.org/mediawiki/igem.org/e/ef/TMUDoor_project.png"><br />
<br><br />
</div><br />
<div class="clear"></div><br />
<br />
<div class="container_12"><br />
<div class="grid_2"><br />
<p><br></p><br />
</div><br />
<br />
<div class="grid_10"><br />
<br><br />
<ul><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Idea">Idea</a></p></li><br />
<p>Our fundamental idea and background are written here.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/NEWS">Breakthrough</a></li></p><br />
<p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device">Pythagorean_Device</a></li></p><br />
<p></p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Parts">Parts</a></li></p><br />
<p>You find what part we submitted.</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Future_Plan">Future plan</a></li></p><br />
<p>You can see the design of a more complicated device</p><br><br />
<li><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Reference">Reference</a></li></p><br />
<p></p><br><br />
</ul><br />
<br />
</div></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/ResultTeam:TMU-Tokyo/Notebook experiment/Result2013-09-28T03:22:51Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/notebook.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<html><br />
<style><br />
.LB{margin-left:105px;<br />
margin-right:auto;}<br />
img.work {display:block;<br />
margin-left:auto;<br />
margin-right:auto;<br />
}<br />
</style><br />
<br />
<br><br />
<br><br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Result</h1><hr><br />
<br><br />
<br><br />
<div class="LB"><br />
<img src="https://static.igem.org/mediawiki/2013/f/fa/TMUResult-LB.png"><img width="250"height="256"src="https://static.igem.org/mediawiki/2013/7/77/TMUResult-ara.png"><img width="250"height="256" src="https://static.igem.org/mediawiki/2013/d/d6/TMUResult-IPTG.png"><br />
</div><br />
<br><br />
<br><br />
<img class="work" src="https://static.igem.org/mediawiki/2013/9/9e/TMUresult-PCR0928.png"><br />
<br />
<br><br />
<p></p><br />
<br />
<br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Notebook_experiment/ResultTeam:TMU-Tokyo/Notebook experiment/Result2013-09-28T03:18:52Z<p>Masashiohara: </p>
<hr />
<div>{{Template:TMU-Tokyo/css/reset.css}}<br />
{{Template:Team:TMU-Tokyo/header}}<br />
{{Template:Team:TMU-Tokyo/notebook.css}}<br />
{{Template:Team:TMU-Tokyo/drop-menu.css}}<br />
{{Template:Team:TMU-Tokyo/css/960.css}}<br />
<html><br />
<style><br />
.LB{margin-left:105px;<br />
margin-right:auto;}<br />
<br />
<br />
</style><br />
<br />
<br><br />
<br><br />
<div class="container_12"><br />
<div class="grid_12"><br />
<h1>Result</h1><hr><br />
<br><br />
<br><br />
<div class="LB"><br />
<img src="https://static.igem.org/mediawiki/2013/f/fa/TMUResult-LB.png"><img width="250"height="256"src="https://static.igem.org/mediawiki/2013/7/77/TMUResult-ara.png"><img width="250"height="256" src="https://static.igem.org/mediawiki/2013/d/d6/TMUResult-IPTG.png"><br />
</div><br />
<img src="https://static.igem.org/mediawiki/2013/9/9e/TMUresult-PCR0928.png"><br />
<br />
<br><br />
<p></p><br />
<br />
<br />
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<h1>Result</h1><br />
<Hr><br />
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<img src="https://static.igem.org/mediawiki/2013/f/fa/TMUResult-LB.png"><img src="https://static.igem.org/mediawiki/2013/7/77/TMUResult-ara.png"><img src="https://static.igem.org/mediawiki/2013/d/d6/TMUResult-IPTG.png"><br />
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<img src"https://static.igem.org/mediawiki/2013/f/fa/TMUResult-LB.png"><img src"https://static.igem.org/mediawiki/2013/7/77/TMUResult-ara.png"><img src"https://static.igem.org/mediawiki/2013/d/d6/TMUResult-IPTG.png"><br />
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<div></div>Masashioharahttp://2013.igem.org/File:TMUResult-ara.pngFile:TMUResult-ara.png2013-09-28T03:00:59Z<p>Masashiohara: </p>
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<div></div>Masashioharahttp://2013.igem.org/File:TMUResult-LB.pngFile:TMUResult-LB.png2013-09-28T03:00:32Z<p>Masashiohara: </p>
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<div></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T02:58:30Z<p>Masashiohara: </p>
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<h1>Safty Check List</h1><br />
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<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
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<h1>Safety Guidance by Teachers</h1><br />
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<table><br />
<tr><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Phytohormone Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<div class="grid_12"><br />
<br><br />
<br><br />
<p><br><br />
We thought that to experiment safely is very important like we wrote “iGEM 2013 basic safety form.” Not safe experiments are meaningless. We must protect biodiversity against genetically modified organisms (GMOs). Also we must not impact badly on our body.<br><br />
Therefore, we received our teacher’s guidance to experiment safely at a laboratory. In the guidance, we are taught matters to be attended to experiment, operation procedures of experimental devices and laboratory and treatment of dangerous objects.<br><br />
We’ve experimented along their lab’s rule.<br><br />
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<h1>Submitted parts</h1><br />
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<center><groupparts>iGEM013 TMU-Tokyo</groupparts></center><br />
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<h1>Parts data</h1><br />
<hr><br />
<span style="line-height:50%"><Br></span><br />
<ol><br />
<li><a href="http://parts.igem.org/Part:BBa_K1015013">BBa_K1015013</a>:<b>Km-Blunt end </b><br><br />
This is what connected the blunt end to cohesive end of pSB1C3.</li><br />
<span style="line-height:50%"><Br></span><br />
<li><a href="http://parts.igem.org/Part:BBa_K1015014">BBa_K1015014</a>:<b>IRR/IRL(Ptet)-FLP-Sm(insert between lacA and cynR)</b><br><br />
This device produce FLP and AadA(streptomycin resistant gene) . But if site specific recombination be caused between IRR site and IRL site by FimE recombinase, pTet promoter change "OFF" state and FLP production stop. <br />
</li><br />
<span style="line-height:50%"><Br></span><br />
<li><a href="http://parts.igem.org/Part:BBa_K1015015">BBa_K1015015</a>:<b>araC-pBAD-hbiF-Tc(genome insertion parts between araC and araA)</b><br><br />
With bacteriophage λ recombination system, this device insert into Escherichia coli genome. This device produce hbiF recombinase and tetracycline resistance protein (tetA). HbiF production level are regulated by arabinose inducible promoter pBAD.</li><br />
<span style="line-height:50%"><Br></span><br />
<li><a href="http://parts.igem.org/Part:BBa_K1015016">BBa_K1015016</a>:<b>lacI+FRT+Cm</b><br><br />
This is genome insertion part. The device is: R(SaApR)homology sequence(BBa_K1015018) + lacI(BBa_K1015008) + FRT(BBa_K1015009) + mhpA homology sequence(BBa_K1015009).<br />
</li> <br />
<span style="line-height:50%"><Br></span><br />
<li><a href="http://parts.igem.org/Part:BBa_K1015017">BBa_K1015017</a>:<b>FRT+Ap(for RED system)</b><br><br />
This is genome insertion parts. Site specific recombination site. if it happens by Flp sequences between 2 place of FRT is left out. </li><br />
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<h1>Project description<h1><br />
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<td><p>In the iGEM, most teams have inserted their Biobrick parts or devices into plasmids and have used them. It is true that there are many good points to use plasmids, for instance, it is easy to do transformation, and it is convenient to use high copy plasmid when you want to get high amount of gene expression. However, there are some bad points to use plasmids too. For example, when the reproduction starting point of plural plasmids are covered, they can’t be put in <i>E.coli</i> at the same time. Also it is difficult to control closely expression of the genes which are in .plasmids.</p><br />
<p>Therefore, in this year, our team tried to establish and standardize a new method to insert Biobrick parts or devices in a genome of <i>E.coli</i> and use them. Also, according to this method, we really inserted the device which we designed in a genome of <i>E.coli</i> and functionalized it.</p><br />
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<tr height="30"><td align="center"><b>Standardization</b> -New method, “Breakthrough”-</td></tr><br />
<tr height="200"><td style="padding:3px 7px;"><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/NEWS" >In order to insert Biobricks in a genome of <i>E.coli</i> and functionalize them, we established a new and interesting method and standardize it. In this new method named “Breakthrough”, we constructed all of our parts by over rap extension PCR and inserted these PCR products into a genome of <i>E.coli</i> by lambda bacteriophage recombination system named ”RED”. Also we designed a new parts which to improve the present designated vector (pSB1C3). With this part, it can be easily to do cloning the parts which made by PCR.<br><br />
Additionally, we standardized this method so that other iGEMers could use it. So<u> we’ll apply for New Standard prize.</u><br />
</p><br />
</td></tr><br />
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</td><br />
<td align="center"><br />
<table class="imple" rules="none" border="1" width="450" height="270" "><br />
<tr height="30"><td align="center"><b>Implementation</b> ―Genomic Pythagorean Device-</a></td></tr><br />
<br />
<tr height="200"><td style="padding:3px 7px;"><br />
<p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device" > According to the method that we spoke in the right, we really inserted the device named “Genomic Pythagorean Device”, which we designed into a genome of <i>E.coli</i>. “Pythagorean Device” is very famous in Japan because Japanese famous educational TV program "Pythagorean Switch" introduces various Pythagorean Devices. They are known as "Rube Goldberg machines" in the US. Pythagorean Devices are deliberately over-engineered or overdone systems that perform very simple task in very complicated process, and they usually include some chain reactions. We constructed “Pythagorean Device” in a genome of <i>E.coli</i> and checked whether it functioned properly.</a></p></td></tr><br />
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<box class="idea"><br />
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<td class="boximg" rowspan="2" ><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Idea"><br />
<img src="https://static.igem.org/mediawiki/igem.org/9/9b/TMU-Idea.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/e/e5/TMU-Idea_af.png';"<br />
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<td colspan="2" rowspan="2"><br />
<li>What is our goal?</li> <br />
<li>Why is there a need for our project?</li> <br />
<li>Why do we insert parts in a genome? </td><br />
</tr><br />
<tr><br />
</tr><br />
<tr><br />
<td ></td><br />
<td></td><br />
<td></td><br />
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</td><br />
<td align="center"><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Parts"><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/0c/TMUParts.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/2/2e/TMUParts_af.png';"<br />
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<td colspan="2" rowspan="2"><br />
<li>What Biobrick parts did we design ?</li><br />
<li>Which parts did we submit ?</li><br />
<li>Did our parts work ?</li> </td><br />
</tr><br />
<tr><br />
</tr><br />
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<td ></td><br />
<td></td><br />
<td></td><br />
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</td><br />
</tr><br />
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<box class="future"><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Future_Plan"><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/11/TMUArrow.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/f/fe/TMUArrow_af.png';"<br />
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<td colspan="2" rowspan="2"><br />
<li>What kind of application is imaginable ?</li><br />
<li>What is necessary to improve our project ?</li><br />
</td><br />
</tr><br />
<tr><br />
</tr><br />
<tr><br />
<td ></td><br />
<td></td><br />
<td></td><br />
</tr><br />
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</td><br />
<td align="center"> <br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/HumanPractice"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/c8/TMUHeart.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/3/32/TMUHeart_af.png';"<br />
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<td colspan="2" rowspan="2"><br />
<li>What did we do to promote Synthetic Biology?</li><br />
</td><br />
</tr><br />
<tr><br />
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<td ></td><br />
<td></td><br />
<td></td><br />
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</td><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety"><br />
<img src="https://static.igem.org/mediawiki/igem.org/2/23/TMUSafety.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/5/5a/TMUSafety_af.png';"<br />
onmouseout="this.src='https://static.igem.org/mediawiki/igem.org/2/23/TMUSafety.png';"></a></td><br />
<td colspan="2" rowspan="2"><br />
<li>What did we do for securing of safety ?</li><br />
</td><br />
</tr><br />
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</tr><br />
<tr><br />
<td ></td><br />
<td></td><br />
<td></td><br />
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</td><br />
<td align="center"> <br />
<box class="achievement"><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Achievement"><br />
<img src="https://static.igem.org/mediawiki/igem.org/5/52/TMUTrophy.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/a/a2/TMUTrophy_af.png';"<br />
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<td colspan="2" rowspan="2"><br />
<li>What did we achieve in our project ?</li><br />
</td><br />
</tr><br />
<tr><br />
</tr><br />
<tr><br />
<td ></td><br />
<td></td><br />
<td></td><br />
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</td><br />
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<h1>Sponsors</h1><br />
<Hr><br />
<Br><br />
<A href="http://www.promega.co.jp/index.html"><IMg src="https://static.igem.org/mediawiki/2013/f/f9/Promega_logo_c.gif"></A><br />
<A href="http://lne.st"><IMG Src="https://static.igem.org/mediawiki/2013/8/89/Lnest-logo3_03.png" border="0"height="60"><br />
</A><br />
<A href="http://mendel-science.com/"><IMG Src="http://igemtmu.net/2011/menderu.png" border="0"><br />
</A><br />
<br />
<A href="http://www.cosmobio.co.jp"><IMG Src="http://igemtmu.net/2011/kosumo.png" border="0"><br />
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<br />
<A href="http://www.mbl.co.jp/"><IMG Src="https://static.igem.org/mediawiki/2012/f/f2/Mbl.gif" border="0"><br />
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</A><br />
<br />
<A href="http://www.tmu.ac.jp/"><IMG Src="https://static.igem.org/mediawiki/2012/c/c6/TMUlogo.png" border="0"><br />
</A><br />
<A href="http://www.comp.tmu.ac.jp/dousoukai/"><IMG Src="https://static.igem.org/mediawiki/2012/e/e1/Dousoukai.sp.jpg" border="0"><br />
<a href="http://www.ikedarika.co.jp/"><img src="https://static.igem.org/mediawiki/igem.org/c/c2/Ikeda_logo.gif" ><br />
<Br><br />
<Br><br />
<br />
</A><br />
</p><br />
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<div class="grid_3"><br />
</div><br />
<h1>contact us!!</h1><br />
<Hr><br />
<Br><br />
<br />
<p align="center" hspace="20"><a href="https://www.facebook.com/TmUnigem"><img src="https://static.igem.org/mediawiki/igem.org/6/66/TMUFacebook02.jpg" ></a></p><br />
<span style="line-height:50%"><Br></span> <br />
<p><img vspace="20" src="https://static.igem.org/mediawiki/igem.org/5/50/TMUGmail-Icon.png">tmutokyo.igem@gmail.com</p><br />
<br />
<br />
<br />
</div><br />
</div><br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/Future_PlanTeam:TMU-Tokyo/Project/Future Plan2013-09-28T02:48:44Z<p>Masashiohara: </p>
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<h1>More improved “Genomic Pythagorean Device”</h1><br />
<hr><br />
<br><br />
<p><u></u></p><br />
<br><br />
<p>We designed three kinds of devices which improved “Genomic Pythagorean Device”. The basic fragments constituting these three devices are same with the original device (Fig.1).</p><br />
<br />
<img class="plan" width="520" height="385" src="https://static.igem.org/mediawiki/2013/1/17/TMUFig.1.png"><br />
<br><br />
<p>If you want to see more detailed explanation of our device which we made in this year, please go and look<a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device"> “Genomic Pythagorean Device"</a>page.</p><br />
<br />
<br><br />
<br><br />
<p>・Improved device 1</p><br />
<p>In this year, we did PCR in order to check that lacI gene is deleted because of the presence of FRT. It was a good assay method but it takes time slightly. So first we designed the improved device 1 which is for easily assay of fragment 3-1 and 3-2. This device has GFP site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off. Then GFP comes to be expressed so that we can confirm easily whether lacI gene is deleted or not. (Fig.2)</p><br />
<br><br />
<img class="plan" width="640" height="320"src="https://static.igem.org/mediawiki/2013/b/b1/TMUFuture.fig2.png"><br />
<br />
<br><br />
<br><br />
<br />
<p>・Improved device 2</p><br />
<br />
<p>We designed more complicated device by using the site of lox66, lox71 and Cre recombinase. Lox66 and lox71 are two mutants of loxP site and Cre recombinase mediated recombination between them. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase. So once Cre-mediated recombination happen, it becomes difficult to happen again. (Fig.3)</p><br />
<br><br />
<img class="plan" width="640" height="400"src="https://static.igem.org/mediawiki/2013/7/77/TMUFuture.fig3.png"><br />
<br />
<br><br />
<p>Then, we’ll describe that how this device works. This device has Cre recominase site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off and Cre recombinase comes to be expressed. Then site specific recombination happen between the sites of lox66 and lox71 and the promoter which is in there turns over by inversion. By this result, GFP comes to be expressed and we can confirm easily whether lacI gene is deleted or not (Fig.4).</p><br />
<img class="plan"width="640" height="520" src="https://static.igem.org/mediawiki/2013/9/9c/TMUFuture.fig4.png"><br />
<br><br />
<br><br />
<br />
<p>・Improved device 3</p><br />
<p>We designed more complicated device than improved device 2 by using attB/attP recombination system in addition of lox66, lox71 and Cre recombinase. Site specific recombinase named Int/Xis mediate recombination between the sites of attB and attP.</p><br />
<p>This device has Cre recominase site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off and Cre recombinase comes to be expressed. Then site specific recombination happen between the sites of lox66 and lox71 and the promoter which is in there turns over by inversion. By this result, Int comes to be expressed. Then, site specific recombination happen because of the presence of Int and the plasmid which has the site of loxP and CI repressor is inserted in E.coli genome. CI repress the activity of pCI. Then Cre recombinase mediates the site specific recombination between the 2sites of loxP and CI repressor gene is deleted. By this result, repression of pCI becomes off and GFP comes to be expressed. (Fig.5)</p><br />
<img class="plan" width="800" height="1200"src="https://static.igem.org/mediawiki/2013/b/b3/TMUFuture.fig5.png"><br />
<br><br />
<br><br />
<br><br />
<h1>"Genomic Kill Switch"</h1><br />
<hr><br />
<p>In Biobricks that are inserted in genome, horizontal gene transfer rate is lower than <br />
Biobricks are shipped by plasmids. This point is very important merit at the aspect of <br />
biosafety.</p><br />
<p>However, BioBricks which are in genome have stable hereditary too. This point may <br />
become demerit at the aspect of biosafety. If the organisms which have some BioBricks<br />
are in their genome released to natural environment, genetically modified materials would <br />
remain stably in natural environment.</p><br />
<p>In order to solve these problems, we designed “Genomic Kill Switch” as our future plan. <br />
The switch changes E.coli into arabinose requirement.</p><br />
<p>This device has bacteriophage p22 cII repressor in the downstream of pBAD promoter<br />
which is activated by L-arabinose. And BBa_K124017 (which has Bacteriophage Lysis <br />
Cassette S105, R, and Rz) are placed in the downstream of p22 c II regulated promoter.<br />
According to this device, BBa_K124017 expression is repressed by p22 cII only when <br />
arabinose presents in environment. It is really difficult to maintain this condition in natural <br />
environment, so if the E.coli which have the Genomic kill switch released by accident, they <br />
can’t live any longer. Therefore, this switch is able to prevent to spread genetically modified<br />
materials.</p><br />
<img class="plan" width="720" height="520"src="https://static.igem.org/mediawiki/2013/a/a0/TMUFuture.kill1.png"><br />
<img class="plan" width="720" height="520"src="https://static.igem.org/mediawiki/2013/7/70/TMUFuture.kill2.png"><br />
<br />
<br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/NEWSTeam:TMU-Tokyo/Project/NEWS2013-09-28T02:42:04Z<p>Masashiohara: </p>
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<h1>How to insert fragments in E.coli gonome</h1><br />
<hr><br />
<br><br />
<br><br />
<img class="work"width="768" height="960" src="https://static.igem.org/mediawiki/2013/6/62/How_to_insert_1_TMU.png"><br />
<img class="work"width="768" height="960" src="https://static.igem.org/mediawiki/2013/0/04/TMUNEWS.c.png"><br />
<img class="work" width="768" height="360" src="https://static.igem.org/mediawiki/2013/4/44/TMUNEWS.d.png"><br />
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<br><br />
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<br><br />
</div><br />
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<h1>Instruction Manual of this method</h1><br />
<hr><br />
<br><br />
<img class="work"width="640"height="632" src="https://static.igem.org/mediawiki/igem.org/2/28/Breakthrough_2_TMU.png"><br />
<br><br />
<p> We’ll explain how you should use this method using an example of <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></p><br />
<br />
<br><br />
<p><u>At the beginning…</u></p><br />
<p>You should choose two non-essential genes from the list.<br />
It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).<br />
</p><br />
<br><br />
<br><br />
<br />
<h5 style="color:red;">Attention</h5><br />
<p>You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)</p><br />
<br><br />
<br />
<br><br />
<p>1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers.<br />
By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.<br />
</p><br />
<br />
<p><Primer list></p><br />
<p>Primer 1 : Cm primer (forward)</p><br />
<p>Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)</p><br />
<p>Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2</p><br />
<p>Primer 4 : VR + homology site of Cm resistant gene</p><br />
<br><br />
<br><br />
<br />
<p>2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of BBa_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.</p><br />
<br><br />
<p> Primer of yahA (from gene list)</p><br />
<p>Primer of yahC (from gene list) </p><br />
<br><br />
<br><br />
<br />
<p>3. Next, you should make the following insertion fragment, “yahA homology site+BBa_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.<br />
</p><br />
<br><br />
<p>4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.<br><br />
<br><br />
<br><br />
You should try this method!</p><br />
<br />
<br><br />
<div style="font-size:26px;">You can download <a href="https://2013.igem.org/File:TMUNew_standard_2.xls">Primer list</a>.</div><br />
<br><br />
<br />
</div><br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/NEWSTeam:TMU-Tokyo/Project/NEWS2013-09-28T02:24:44Z<p>Masashiohara: </p>
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<h1>How to insert fragments in E.coli gonome</h1><br />
<hr><br />
<br><br />
<br><br />
<img class="work"width="768" height="960" src="https://static.igem.org/mediawiki/2013/6/62/How_to_insert_1_TMU.png"><br />
<img cwidth="768" height="960" src="https://static.igem.org/mediawiki/2013/0/04/TMUNEWS.c.png"><br />
<img class="work" width="768" height="360" src="https://static.igem.org/mediawiki/2013/4/44/TMUNEWS.d.png"><br />
<br />
<br><br />
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<br><br />
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<div class="container_12"><br />
<div class="grid_12"><br />
<br />
<h1>Instruction Manual of this method</h1><br />
<hr><br />
<br><br />
<img class="work"width="1000"height="800" src="https://static.igem.org/mediawiki/igem.org/2/28/Breakthrough_2_TMU.png"><br />
<br><br />
<p> We’ll explain how you should use this method using an example of <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></p><br />
<br />
<br><br />
<p><u>At the beginning…</u></p><br />
<p>You should choose two non-essential genes from the list.<br />
It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).<br />
</p><br />
<br><br />
<br><br />
<br />
<h5 style="color:red;">Attention</h5><br />
<p>You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)</p><br />
<br><br />
<br />
<br><br />
<p>1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers.<br />
By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.<br />
</p><br />
<br />
<p><Primer list></p><br />
<p>Primer 1 : Cm primer (forward)</p><br />
<p>Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)</p><br />
<p>Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2</p><br />
<p>Primer 4 : VR + homology site of Cm resistant gene</p><br />
<br><br />
<br><br />
<br />
<p>2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of BBa_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.</p><br />
<br><br />
<p> Primer of yahA (from gene list)</p><br />
<p>Primer of yahC (from gene list) </p><br />
<br><br />
<br><br />
<br />
<p>3. Next, you should make the following insertion fragment, “yahA homology site+BBa_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.<br />
</p><br />
<br><br />
<p>4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.<br><br />
<br><br />
<br><br />
You should try this method!</p><br />
<br />
<br><br />
<div style="font-size:26px;">You can download <a href="https://2013.igem.org/File:TMUNew_standard_2.xls">Primer list</a>.</div><br />
<br><br />
<br />
</div><br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/NEWSTeam:TMU-Tokyo/Project/NEWS2013-09-28T02:24:00Z<p>Masashiohara: </p>
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<div class="grid_12"><br />
<h1>How to insert fragments in ''E.coli'' gonome</h1><br />
<hr><br />
<br><br />
<br><br />
<img class="work"width="768" height="960" src="https://static.igem.org/mediawiki/2013/6/62/How_to_insert_1_TMU.png"><br />
<img cwidth="768" height="960" src="https://static.igem.org/mediawiki/2013/0/04/TMUNEWS.c.png"><br />
<img class="work" width="768" height="360" src="https://static.igem.org/mediawiki/2013/4/44/TMUNEWS.d.png"><br />
<br />
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<br><br />
</div><br />
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<br />
<h1>Instruction Manual of this method</h1><br />
<hr><br />
<br><br />
<img class="work"width="1000"height="800" src="https://static.igem.org/mediawiki/igem.org/2/28/Breakthrough_2_TMU.png"><br />
<br><br />
<p> We’ll explain how you should use this method using an example of <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></p><br />
<br />
<br><br />
<p><u>At the beginning…</u></p><br />
<p>You should choose two non-essential genes from the list.<br />
It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).<br />
</p><br />
<br><br />
<br><br />
<br />
<h5 style="color:red;">Attention</h5><br />
<p>You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)</p><br />
<br><br />
<br />
<br><br />
<p>1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers.<br />
By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.<br />
</p><br />
<br />
<p><Primer list></p><br />
<p>Primer 1 : Cm primer (forward)</p><br />
<p>Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)</p><br />
<p>Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2</p><br />
<p>Primer 4 : VR + homology site of Cm resistant gene</p><br />
<br><br />
<br><br />
<br />
<p>2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of BBa_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.</p><br />
<br><br />
<p> Primer of yahA (from gene list)</p><br />
<p>Primer of yahC (from gene list) </p><br />
<br><br />
<br><br />
<br />
<p>3. Next, you should make the following insertion fragment, “yahA homology site+BBa_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.<br />
</p><br />
<br><br />
<p>4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.<br><br />
<br><br />
<br><br />
You should try this method!</p><br />
<br />
<br><br />
<div style="font-size:26px;">You can download <a href="https://2013.igem.org/File:TMUNew_standard_2.xls">Primer list</a>.</div><br />
<br><br />
<br />
</div><br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-TokyoTeam:TMU-Tokyo2013-09-28T02:20:30Z<p>Masashiohara: </p>
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<h1>Project description<h1><br />
<Hr><br />
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<td><p>In the iGEM, most teams have inserted their Biobrick parts or devices into plasmids and have used them. It is true that there are many good points to use plasmids, for instance, it is easy to do transformation, and it is convenient to use high copy plasmid when you want to get high amount of gene expression. However, there are some bad points to use plasmids too. For example, when the reproduction starting point of plural plasmids are covered, they can’t be put in <i>E.coli</i> at the same time. Also it is difficult to control closely expression of the genes which are in .plasmids.</p><br />
<p>Therefore, in this year, our team tried to establish and standardize a new method to insert Biobrick parts or devices in a genome of <i>E.coli</i> and use them. Also, according to this method, we really inserted the device which we designed in a genome of <i>E.coli</i> and functionalized it.</p><br />
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<tr height="30"><td align="center"><b>Standardization</b> -New method, “Breakthrough”-</td></tr><br />
<tr height="200"><td style="padding:3px 7px;"><p><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/NEWS" >In order to insert Biobricks in a genome of <i>E.coli</i> and functionalize them, we established a new and interesting method and standardize it. In this new method named “Breakthrough”, we constructed all of our parts by over rap extension PCR and inserted these PCR products into a genome of <i>E.coli</i> by lambda bacteriophage recombination system named ”RED”. Also we designed a new parts which to improve the present designated vector (pSB1C3). With this part, it can be easily to do cloning the parts which made by PCR.<br><br />
Additionally, we standardized this method so that other iGEMers could use it. So<u> we’ll apply for New Standard prize.</u><br />
</p><br />
</td></tr><br />
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<td align="center"><br />
<table class="imple" rules="none" border="1" width="450" height="270" "><br />
<tr height="30"><td align="center"><b>Implementation</b> ―Genomic Pythagorean Device-</a></td></tr><br />
<br />
<tr height="200"><td style="padding:3px 7px;"><br />
<p><a href="www.google.co.jp" > According to the method that we spoke in the right, we really inserted the device named “Genomic Pythagorean Device”, which we designed into a genome of <i>E.coli</i>. “Pythagorean Device” is very famous in Japan because Japanese famous educational TV program "Pythagorean Switch" introduces various Pythagorean Devices. They are known as "Rube Goldberg machines" in the US. Pythagorean Devices are deliberately over-engineered or overdone systems that perform very simple task in very complicated process, and they usually include some chain reactions. We constructed “Pythagorean Device” in a genome of <i>E.coli</i> and checked whether it functioned properly.</a></p></td></tr><br />
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<box class="idea"><br />
<table border="0" width="450" height="100" bgcolor="#fff"><br />
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<td class="boximg" rowspan="2" ><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Idea"><br />
<img src="https://static.igem.org/mediawiki/igem.org/9/9b/TMU-Idea.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/e/e5/TMU-Idea_af.png';"<br />
onmouseout="this.src='https://static.igem.org/mediawiki/igem.org/9/9b/TMU-Idea.png';"></a></td><br />
<td colspan="2" rowspan="2"><br />
<li>What is our goal?</li> <br />
<li>Why is there a need for our project?</li> <br />
<li>Why do we insert parts in a genome? </td><br />
</tr><br />
<tr><br />
</tr><br />
<tr><br />
<td ></td><br />
<td></td><br />
<td></td><br />
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<td align="center"><br />
<box class="parts"> <br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Parts"><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/0c/TMUParts.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/2/2e/TMUParts_af.png';"<br />
onmouseout="this.src='https://static.igem.org/mediawiki/igem.org/0/0c/TMUParts.png';"></td><br />
<td colspan="2" rowspan="2"><br />
<li>What Biobrick parts did we design ?</li><br />
<li>Which parts did we submit ?</li><br />
<li>Did our parts work ?</li> </td><br />
</tr><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Future_Plan"><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/11/TMUArrow.png"<br />
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<li>What kind of application is imaginable ?</li><br />
<li>What is necessary to improve our project ?</li><br />
</td><br />
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<td ></td><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/HumanPractice"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/c8/TMUHeart.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/3/32/TMUHeart_af.png';"<br />
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<td colspan="2" rowspan="2"><br />
<li>What did we do to promote Synthetic Biology?</li><br />
</td><br />
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<td ></td><br />
<td></td><br />
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<box class="owari"><br />
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<box class="safety"><br />
<table border="0" width="450" height="100" bgcolor="#fff"><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Safety"><br />
<img src="https://static.igem.org/mediawiki/igem.org/2/23/TMUSafety.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/5/5a/TMUSafety_af.png';"<br />
onmouseout="this.src='https://static.igem.org/mediawiki/igem.org/2/23/TMUSafety.png';"></a></td><br />
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<li>What did we do for securing of safety ?</li><br />
</td><br />
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<td ></td><br />
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<box class="achievement"><br />
<table border="0" width="450" height="100" bgcolor="#fff"><br />
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<td class="boximg" rowspan="2"><a href="https://2013.igem.org/Team:TMU-Tokyo/Achievement"><br />
<img src="https://static.igem.org/mediawiki/igem.org/5/52/TMUTrophy.png"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/igem.org/a/a2/TMUTrophy_af.png';"<br />
onmouseout="this.src='https://static.igem.org/mediawiki/igem.org/5/52/TMUTrophy.png';"></a></td><br />
<td colspan="2" rowspan="2"><br />
<li>What did we achieve in our project ?</li><br />
</td><br />
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<td ></td><br />
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<div class="grid_9"><br />
<br />
<h1>Sponsors</h1><br />
<Hr><br />
<Br><br />
<A href="http://www.promega.co.jp/index.html"><IMg src="https://static.igem.org/mediawiki/2013/f/f9/Promega_logo_c.gif"></A><br />
<A href="http://lne.st"><IMG Src="https://static.igem.org/mediawiki/2013/8/89/Lnest-logo3_03.png" border="0"height="60"><br />
</A><br />
<A href="http://mendel-science.com/"><IMG Src="http://igemtmu.net/2011/menderu.png" border="0"><br />
</A><br />
<br />
<A href="http://www.cosmobio.co.jp"><IMG Src="http://igemtmu.net/2011/kosumo.png" border="0"><br />
</A><br />
<br />
<A href="http://www.mbl.co.jp/"><IMG Src="https://static.igem.org/mediawiki/2012/f/f2/Mbl.gif" border="0"><br />
</A><br />
<br />
</A><br />
<br />
<A href="http://www.tmu.ac.jp/"><IMG Src="https://static.igem.org/mediawiki/2012/c/c6/TMUlogo.png" border="0"><br />
</A><br />
<A href="http://www.comp.tmu.ac.jp/dousoukai/"><IMG Src="https://static.igem.org/mediawiki/2012/e/e1/Dousoukai.sp.jpg" border="0"><br />
<a href="http://www.ikedarika.co.jp/"><img src="https://static.igem.org/mediawiki/igem.org/c/c2/Ikeda_logo.gif" ><br />
<Br><br />
<Br><br />
<br />
</A><br />
</p><br />
</div><br />
<div class="grid_3"><br />
</div><br />
<h1>contact us!!</h1><br />
<Hr><br />
<Br><br />
<br />
<p align="center" hspace="20"><a href="https://www.facebook.com/TmUnigem"><img src="https://static.igem.org/mediawiki/igem.org/6/66/TMUFacebook02.jpg" ></a></p><br />
<span style="line-height:50%"><Br></span> <br />
<p><img vspace="20" src="https://static.igem.org/mediawiki/igem.org/5/50/TMUGmail-Icon.png">tmutokyo.igem@gmail.com</p><br />
<br />
<br />
<br />
</div><br />
</div><br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/NEWSTeam:TMU-Tokyo/Project/NEWS2013-09-28T02:20:17Z<p>Masashiohara: </p>
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<h1>“Breakthrough”</h1><br />
<hr><br />
<br><br />
<br><br />
<img class="work"width="768" height="960" src="https://static.igem.org/mediawiki/2013/6/62/How_to_insert_1_TMU.png"><br />
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<h1>Instruction Manual of this method</h1><br />
<hr><br />
<br><br />
<img class="work"width="1000"height="800" src="https://static.igem.org/mediawiki/igem.org/2/28/Breakthrough_2_TMU.png"><br />
<br><br />
<p> We’ll explain how you should use this method using an example of <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></p><br />
<br />
<br><br />
<p><u>At the beginning…</u></p><br />
<p>You should choose two non-essential genes from the list.<br />
It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).<br />
</p><br />
<br><br />
<br><br />
<br />
<h5 style="color:red;">Attention</h5><br />
<p>You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)</p><br />
<br><br />
<br />
<br><br />
<p>1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers.<br />
By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.<br />
</p><br />
<br />
<p><Primer list></p><br />
<p>Primer 1 : Cm primer (forward)</p><br />
<p>Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)</p><br />
<p>Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2</p><br />
<p>Primer 4 : VR + homology site of Cm resistant gene</p><br />
<br><br />
<br><br />
<br />
<p>2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of BBa_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.</p><br />
<br><br />
<p> Primer of yahA (from gene list)</p><br />
<p>Primer of yahC (from gene list) </p><br />
<br><br />
<br><br />
<br />
<p>3. Next, you should make the following insertion fragment, “yahA homology site+BBa_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.<br />
</p><br />
<br><br />
<p>4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.<br><br />
<br><br />
<br><br />
You should try this method!</p><br />
<br />
<br><br />
<div style="font-size:26px;">You can download <a href="https://2013.igem.org/File:TMUNew_standard_2.xls">Primer list</a>.</div><br />
<br><br />
<br />
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</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/Future_PlanTeam:TMU-Tokyo/Project/Future Plan2013-09-28T02:15:30Z<p>Masashiohara: </p>
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<h1>More improved “Genomic Pythagorean Device”</h1><br />
<hr><br />
<br><br />
<p><u></u></p><br />
<br><br />
<p>We designed three kinds of devices which improved “Genomic Pythagorean Device”. The basic fragments constituting these three devices are same with the original device (Fig.1).</p><br />
<br />
<img class="plan" width="520" height="385" src="https://static.igem.org/mediawiki/2013/1/17/TMUFig.1.png"><br />
<br><br />
<p>If you want to see more detailed explanation of our device which we made in this year, please go and look<a href="https://2013.igem.org/Team:TMU-Tokyo/Project/Pythagorean_Device"> “Genomic Pythagorean Device"</a>page.</p><br />
<br />
<br><br />
<br><br />
<p>・Improved device 1</p><br />
<p>In this year, we did PCR in order to check that lacI gene is deleted because of the presence of FRT. It was a good assay method but it takes time slightly. So first we designed the improved device 1 which is for easily assay of fragment 3-1 and 3-2. This device has GFP site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off. Then GFP comes to be expressed so that we can confirm easily whether lacI gene is deleted or not. (Fig.2)</p><br />
<br><br />
<img class="plan" width="640" height="320"src="https://static.igem.org/mediawiki/2013/b/b1/TMUFuture.fig2.png"><br />
<br />
<br><br />
<br><br />
<br />
<p>・Improved device 2</p><br />
<br />
<p>We designed more complicated device by using the site of lox66, lox71 and Cre recombinase. Lox66 and lox71 are two mutants of loxP site and Cre recombinase mediated recombination between them. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase. So once Cre-mediated recombination happen, it becomes difficult to happen again. (Fig.3)</p><br />
<br><br />
<img class="plan" width="640" height="400"src="https://static.igem.org/mediawiki/2013/7/77/TMUFuture.fig3.png"><br />
<br />
<br><br />
<p>Then, we’ll describe that how this device works. This device has Cre recominase site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off and Cre recombinase comes to be expressed. Then site specific recombination happen between the sites of lox66 and lox71 and the promoter which is in there turns over by inversion. By this result, GFP comes to be expressed and we can confirm easily whether lacI gene is deleted or not (Fig.4).</p><br />
<img class="plan"width="640" height="520" src="https://static.igem.org/mediawiki/2013/9/9c/TMUFuture.fig4.png"><br />
<br><br />
<br><br />
<br />
<p>・Improved device 3</p><br />
<p>We designed more complicated device than improved device 2 by using attB/attP recombination system in addition of lox66, lox71 and Cre recombinase. Site specific recombinase named Int/Xis mediate recombination between the sites of attB and attP.</p><br />
<p>This device has Cre recominase site in the downstream of plac. After lacI gene is deleted, repression of plac becomes off and Cre recombinase comes to be expressed. Then site specific recombination happen between the sites of lox66 and lox71 and the promoter which is in there turns over by inversion. By this result, Int comes to be expressed. Then, site specific recombination happen because of the presence of Int and the plasmid which has the site of loxP and CI repressor is inserted in E.coli genome. CI repress the activity of pCI. Then Cre recombinase mediates the site specific recombination between the 2sites of loxP and CI repressor gene is deleted. By this result, repression of pCI becomes off and GFP comes to be expressed. (Fig.5)</p><br />
<img class="plan" width="800" height="1200"src="https://static.igem.org/mediawiki/2013/b/b3/TMUFuture.fig5.png"><br />
<br><br />
<br><br />
<br><br />
<p>"Genomic Kill Switch"</p><br />
<p>In Biobricks that are inserted in genome, horizontal gene transfer rate is lower than <br />
Biobricks are shipped by plasmids. This point is very important merit at the aspect of <br />
biosafety.</p><br />
<p>However, BioBricks which are in genome have stable hereditary too. This point may <br />
become demerit at the aspect of biosafety. If the organisms which have some BioBricks<br />
are in their genome released to natural environment, genetically modified materials would <br />
remain stably in natural environment.</p><br />
<p>In order to solve these problems, we designed “Genomic Kill Switch” as our future plan. <br />
The switch changes E.coli into arabinose requirement.</p><br />
<p>This device has bacteriophage p22 cII repressor in the downstream of pBAD promoter<br />
which is activated by L-arabinose. And BBa_K124017 (which has Bacteriophage Lysis <br />
Cassette S105, R, and Rz) are placed in the downstream of p22 c II regulated promoter.<br />
According to this device, BBa_K124017 expression is repressed by p22 cII only when <br />
arabinose presents in environment. It is really difficult to maintain this condition in natural <br />
environment, so if the E.coli which have the Genomic kill switch released by accident, they <br />
can’t live any longer. Therefore, this switch is able to prevent to spread genetically modified<br />
materials.</p><br />
<img class="plan" width="720" height="520"src="https://static.igem.org/mediawiki/2013/a/a0/TMUFuture.kill1.png"><br />
<img class="plan" width="720" height="520"src="https://static.igem.org/mediawiki/2013/7/70/TMUFuture.kill2.png"><br />
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</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Team_TeamTeam:TMU-Tokyo/Team Team2013-09-28T02:13:47Z<p>Masashiohara: </p>
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<img style="margin:0 20px 0 30px;" src="https://static.igem.org/mediawiki/igem.org/7/7b/TMU-minibuilding.jpg" width="300" height="200"><br />
<span style="line-height:50%"><Br></span><br />
<p><a href="https://2013.igem.org/Team:TMU-Tokyo/Team" >members</a><br><br />
<p>You could see the information of our team members and instructors.If you put on your mouse pointer on images, you could find some individual profiles.</p><br />
<br />
<br />
</div><br />
<br />
<div class="grid_5"> <br />
<img style="margin:0 20px 0 30px;" src="https://static.igem.org/mediawiki/igem.org/0/09/TMUcollabo.jpg" width="300" height="200"><br />
<span style="line-height:50%"><Br></span><br />
<p><a href="https://2013.igem.org/Team:TMU-Tokyo/Team_attribution">attribution</a><br><br />
<p>Here are the information of our university. Also there are some information about a lot of supports we received from others in this page. </p><br />
<br />
<Br><br />
<Br><br />
<Br><br />
</div> <br />
<br />
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</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Team_TeamTeam:TMU-Tokyo/Team Team2013-09-28T02:13:10Z<p>Masashiohara: </p>
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<span style="line-height:50%"><Br></span><br />
<p><a href="https://2013.igem.org/Team:TMU-Tokyo/Team" >members</a><br><br />
<p>You could see the information of our team members and instructors.If you put on your mouse pointer on images, you could find some individual profiles.</p><br />
<br />
<br />
</div><br />
<br />
<div class="grid_5"> <br />
<img style="margin:0 20px 0 30px;" src="https://static.igem.org/mediawiki/igem.org/0/09/TMUcollabo.jpg" width="300" height="200"><br />
<span style="line-height:50%"><Br></span><br />
<p><a href="https://2013.igem.org/Team:TMU-Tokyo/Team_attribution">attribution</a><br><br />
Here are the information of our university. Also there are some information about a lot of supports we received from others in this page. </p><br />
<br />
<Br><br />
<Br><br />
<Br><br />
</div> <br />
<br />
<br />
<br />
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</html></div>Masashioharahttp://2013.igem.org/File:TMUFuture.kill2.pngFile:TMUFuture.kill2.png2013-09-28T02:12:28Z<p>Masashiohara: </p>
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<div></div>Masashioharahttp://2013.igem.org/File:TMUFuture.kill1.pngFile:TMUFuture.kill1.png2013-09-28T02:12:05Z<p>Masashiohara: </p>
<hr />
<div></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/ReferenceTeam:TMU-Tokyo/Project/Reference2013-09-28T02:03:50Z<p>Masashiohara: </p>
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<h1>Reference</h1><br />
<hr><br />
<li><p>Vol. 93, pp. 10090-10093, September 1996<br />
Biochemistry<br><br />
Signal termination in bacterial chemotaxis: CheZ mediates<br />
dephosphorylation of free rather than switch-bound CheY<br />
(signal transduction/response regulator/flagellar switch/phosphatase/cross-linking)<br />
ANAT BREN,</li><br />
<br><br />
<br />
<li><p>Hamer et al. BMC Systems Biology 2010, 4:3<br />
http://www.biomedcentral.com/1752-0509/4/3Deciphering chemotaxis pathways using cross<br />
species comparisons<br />
Rebecca Hamer1,2†,</li><br />
<br> <br />
<li><p>THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 37, Issue of September 11, pp. 24030–24036, 1998<br><br />
Enhanced Binding of Altered H-NS Protein to Flagellar Rotor<br />
Protein FliG Causes Increased Flagellar Rotational Speed and<br />
Hypermotility in Escherichia coli*<br />
(Received for publication, June 1, 1998, and in revised form, June 29, 1998)<br />
Gina M. Donato</li><br />
<br><br />
<li><p>Published: 15 August 2006<br />
BMC Microbiology 2006, 6:72 doi:10.1186/1471-2180-6-72<br />
This article is available from: <a href="http://www.biomedcentral.com/1471-2180/6/72">http://www.biomedcentral.com/1471-2180/6/72</a><br>H-NS controls metabolism and stress tolerance in Escherichia coli<br />
O157:H7 that influence mouse passage<br />
Irfan Erol1,</li><br />
<br><br />
<li><p>Published in final edited form as:<br />
Curr Opin Chem Biol. 2008 August ; 12(4): 389–399. doi:10.1016/j.cbpa.2008.06.015.<br>Exposing Plasmids as the Achilles’ Heel of Drug-Resistant<br />
Bacteria<br />
Julia J. Williams1</li><br />
<br><br />
<li><p>Molecular Microbiology (2002) 45(1), 1–8<br><br />
Bacterial conjugation: a two-step mechanism for<br />
DNA transport:Matxalen Llosa</li><br />
<br><br />
<li><p>The Limits of Reductionism in Medicine:<br />
Could Systems Biology Offer an Alternative?:<br />
Andrew C. Ahn</li><br />
<br><br />
<li><p>Biophysical Journal Volume 96 March 2009 2439–2448<br><br />
Logarithmic Sensing in Escherichia coli Bacterial Chemotaxis<br />
Yevgeniy V. Kalinin,</li><br />
<br><br />
<li><p>FEBS Letters 545 (2003) 86^95<br>Flagellar movement driven by proton translocation:David F. Blair</li><br />
<br><br />
<br></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/HumanPracticeTeam:TMU-Tokyo/HumanPractice2013-09-28T01:54:13Z<p>Masashiohara: </p>
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<img src="https://static.igem.org/mediawiki/igem.org/2/23/TMUattribution.jpg"width="300" height="200"><br />
<p><a href="https://2013.igem.org/Team:TMU-Tokyo/Human_contribution">Collabolation</a></p><br />
<p>Past one years, we had collaborated with many other iGEM teams in Japan or Macquarie_Australia team. We wrote the state of that collaboration .<br />
</p><br />
<br />
<br />
</div><br />
<br />
<div class="grid_5"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/6/6e/TMUPromote.jpg" width="250" height="200"><br />
<p><a href="https://2013.igem.org/Team:TMU-Tokyo/Human_spread">Promote synthetic biology</a></p><br />
<p>We did activities to promote synthetic biology to other peoples.<br />
We wrote the efforts that we did here.</p><br />
<br />
<Br><br />
<Br><br />
<Br><br />
</div> <br />
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</div> <br />
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<div class="clear"></div><br />
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</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Safety/Lab_SafetyTeam:TMU-Tokyo/Safety/Lab Safety2013-09-28T01:31:10Z<p>Masashiohara: </p>
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<h1>Safty Check List</h1><br />
<hr><br />
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<p><br></p><br />
</div><br />
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<p><br><br />
We planned making a self assessment for our experiment. <br><br />
For that, we made Safety Checklist.<br><br />
The safety of our experiment is ensured by this checklist.<br><br />
<br><br />
<br />
→<a href="https://static.igem.org/mediawiki/2013/a/a9/TMUchecklist.pdf" >Safety Check list(PDF)</a> <br />
<br />
</p><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4f/TMUSafty_checklist.jpg" width="360" height="400" > <br />
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<h1>Safety Guidance by Teachers</h1><br />
<hr><br />
<br><br />
<table><br />
<tr><br />
<td>Date</td><br />
<td>Teacher</td><br />
<td>Employee of Experiment</td><br />
</tr><br />
<tr><br />
<td>July 4th</td><br />
<td>Takeshi Kanegae<br />
<br />
(Environmental Response of Plant Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>September 2nd</td><br />
<td>Junichi Kato<br />
<br />
(Molecular Genetics Lab)</td><br />
<td>Kento Tominaga</td><br />
</tr><br />
<tr><br />
<td>July 10th</td><br />
<td>Takashi Okamoto<br />
<br />
(Phytohormone Lab)</td><br />
<td>Eri Sakurai<br><br />
<br />
Marina Ikeno</td><br />
</tr><br />
<tr><br />
<td>July 24th</td><br />
<td>Shin Haruta<br />
<br />
(Environmental Microbiology Lab)</td><br />
<td>Masashi Ohara<br><br />
<br />
Rena Nakajima</td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
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</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/NEWSTeam:TMU-Tokyo/Project/NEWS2013-09-28T01:14:16Z<p>Masashiohara: </p>
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<img cwidth="768" height="960" src="https://static.igem.org/mediawiki/2013/0/04/TMUNEWS.c.png"><br />
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<h1>Instruction Manual of this method</h1><br />
<hr><br />
<br><br />
<img class="work"width="1000"height="800" src="https://static.igem.org/mediawiki/igem.org/2/28/Breakthrough_2_TMU.png"><br />
<br><br />
<p> We’ll explain how you should use this method using an example of <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></p><br />
<br />
<br><br />
<p><u>At the beginning…</u></p><br />
<p>You should choose two non-essential genes from the list.<br />
It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).<br />
</p><br />
<br><br />
<br><br />
<br />
<h5 style="color:red;">Attention</h5><br />
<p>You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)</p><br />
<br><br />
<br />
<br><br />
<p>1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers.<br />
By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.<br />
</p><br />
<br />
<p><Primer list></p><br />
<p>Primer 1 : Cm primer (forward)</p><br />
<p>Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)</p><br />
<p>Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2</p><br />
<p>Primer 4 : VR + homology site of Cm resistant gene</p><br />
<br><br />
<br><br />
<br />
<p>2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of Bba_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.</p><br />
<br><br />
<p> Primer of yahA (from gene list)</p><br />
<p>Primer of yahC (from gene list) </p><br />
<br><br />
<br><br />
<br />
<p>3. Next, you should make the following insertion fragment, “yahA homology site+Bba_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.<br />
</p><br />
<br><br />
<p>4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.<br><br />
<br><br />
<br><br />
You should try this method!</p><br />
<br />
<br><br />
<div style="font-size:26px;">You can download <a href="https://2013.igem.org/File:TMUNew_standard_2.xls">Primer list</a>.</div><br />
<br><br />
<br />
</div><br />
</html></div>Masashioharahttp://2013.igem.org/File:How_to_insert_1_TMU.pngFile:How to insert 1 TMU.png2013-09-28T01:12:45Z<p>Masashiohara: </p>
<hr />
<div></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/NEWSTeam:TMU-Tokyo/Project/NEWS2013-09-28T01:11:25Z<p>Masashiohara: </p>
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<h1>Instruction Manual of this method</h1><br />
<hr><br />
<br><br />
<img class="work"width="1000"height="800" src="https://static.igem.org/mediawiki/igem.org/2/28/Breakthrough_2_TMU.png"><br />
<br><br />
<p> We’ll explain how you should use this method using an example of <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></p><br />
<br />
<br><br />
<p><u>At the beginning…</u></p><br />
<p>You should choose two non-essential genes from the list.<br />
It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).<br />
</p><br />
<br><br />
<br><br />
<br />
<h5 style="color:red;">Attention</h5><br />
<p>You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)</p><br />
<br><br />
<br />
<br><br />
<p>1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers.<br />
By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.<br />
</p><br />
<br />
<p><Primer list></p><br />
<p>Primer 1 : Cm primer (forward)</p><br />
<p>Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)</p><br />
<p>Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2</p><br />
<p>Primer 4 : VR + homology site of Cm resistant gene</p><br />
<br><br />
<br><br />
<br />
<p>2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of Bba_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.</p><br />
<br><br />
<p> Primer of yahA (from gene list)</p><br />
<p>Primer of yahC (from gene list) </p><br />
<br><br />
<br><br />
<br />
<p>3. Next, you should make the following insertion fragment, “yahA homology site+Bba_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.<br />
</p><br />
<br><br />
<p>4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.<br><br />
<br><br />
<br><br />
You should try this method!</p><br />
<br />
<br><br />
<div style="font-size:26px;">You can download <a href="https://2013.igem.org/File:TMUNew_standard_2.xls">Primer list</a>.</div><br />
<br><br />
<br />
</div><br />
</html></div>Masashioharahttp://2013.igem.org/Team:TMU-Tokyo/Project/NEWSTeam:TMU-Tokyo/Project/NEWS2013-09-27T23:02:06Z<p>Masashiohara: </p>
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<img class="work"width="768" height="960" src="https://static.igem.org/mediawiki/2013/0/04/TMUNEWS.c.png"><br />
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<h1>Instruction Manual of this method</h1><br />
<hr><br />
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<img class="work"width="1000"height="800" src="https://static.igem.org/mediawiki/2013/a/a7/TMUExample.png"><br />
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<p> We’ll explain how you should use this method using an example of <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a></p><br />
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<p><u>At the beginning…</u></p><br />
<p>You should choose two non-essential genes from the list.<br />
It is to be desired that two non-essential genes that you chose are in the same distance with device or gene part that you want to insert in E.coli genome (←In this time, it is BBa_J04450).<br />
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<h5 style="color:red;">Attention</h5><br />
<p>You have to pay attention to the direction of non-essential genes you chose. Also you should check whether essential gene is included in between the site of two non-essential genes. You can easy to see the direction of non-essential genes in this wiki!)</p><br />
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<p>1. In the first PCR, you should amplify Cm resistant gene which is in pSB1C3 and Biobrick part of BBa_J04450 by using following primers.<br />
By the first PCR, you should add the homology site of some non-essential gene1 (For instance, yahC) to the reverse side of Cm resistant gene. Also you should add the homology site of some non-essential gene2 (For instance, yahA) to the forward side of Biobrick part and add the homology site of Cm resistant gene to the reverse side of it.<br />
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<p><Primer list></p><br />
<p>Primer 1 : Cm primer (forward)</p><br />
<p>Primer 2 : Cm primer (reverse) + homology site of non-essential gene2 (refer to the list)</p><br />
<p>Primer 3 : homology site of non-essential gene1 (refer to the list) + VF2</p><br />
<p>Primer 4 : VR + homology site of Cm resistant gene</p><br />
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<p>2. Then, you should run second PCR using primer 2 and 3 which are in our gene list. By this PCR, the homology site which was added to Biobrick part of Bba_J04450 and Cm resistant gene are combined in redundant site and you can make the liner fragment.</p><br />
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<p> Primer of yahA (from gene list)</p><br />
<p>Primer of yahC (from gene list) </p><br />
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<p>3. Next, you should make the following insertion fragment, “yahA homology site+Bba_J04450+Cm resistant gene+yahC homology site” by over rap extension PCR. Then you can insert this fragment in E.coli genome.<br />
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<p>4. Next, you have to insert your fragment in MG1655 by electroporation. MG1655 has a lambda phage recombination system, “RED”. Then, homologous recombination will happen between the homology sites are in the fragment and the genome and finally you’ll succeed in inserting your fragment into E.coli genome. This insertion part has Cm resistant gene, so you can choose colonies which have the fragment. This method is easy to use for all of iGEMers and it’s a convenient and powerful tool for using Biobricks more efficiently.<br><br />
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You should try this method!</p><br />
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<div style="font-size:26px;">You can download <a href="https://2013.igem.org/File:TMUNew_standard_2.xls">Primer list</a>.</div><br />
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</html></div>Masashiohara