http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Oyas&year=&month=2012.igem.org - User contributions [en]2024-03-28T10:38:47ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-08-07T13:55:54Z<p>Oyas: </p>
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</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-08-07T13:44:28Z<p>Oyas: </p>
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</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-08-07T13:42:18Z<p>Oyas: </p>
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</div><br />
<div class="sub-heading visible-desktop" id="sub-heading2"><br />
B<span class="sub-heading-small">acterial</span> A<span class="sub-heading-small">nti</span>-C<span class="sub-heading-small">ancer</span>-K<span class="sub-heading-small">amikaze</span><br />
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</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-08-07T13:41:38Z<p>Oyas: </p>
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<div class="heading visible-tablet visible-desktop"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
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</div></div>Oyashttp://2012.igem.org/Team:NTNU_TrondheimTeam:NTNU Trondheim2013-06-16T20:02:03Z<p>Oyas: </p>
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<p>See how we tested our components and what results we got.</p><br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_TrondheimTeam:NTNU Trondheim2013-06-16T19:56:44Z<p>Oyas: </p>
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<strong>Judge looking for an overview of what we have done?</strong><br/> We suggest you start off by reading through the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Achievements</a> section. There you will find an overview of the criteria we have fulfilled with links to the relevant documentation. <br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Click to read more about our project, Bacterial Anti-Cancer Kamikaze. Our vision: Destruction of cancer cells by means of exploding bacteria!</p><br />
</div><br />
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<p>Documentation of the testing of the components in our genetic circuit.</p><br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
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<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
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</li><br />
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<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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<p style="text-align: center;">Countdown to the <a href="https://2012.igem.org/Regions/Europe/Jamboree" target="_blank">European jamboree</a>:<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_TrondheimTeam:NTNU Trondheim2013-06-16T19:55:13Z<p>Oyas: </p>
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<strong>Judge looking for an overview of what we have done?</strong><br/> We suggest you start off by reading through the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Achievements</a> section. There you will find an overview of the criteria we have fulfilled with links to the relevant documentation. <br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Click to read more about our project, Bacterial Anti-Cancer Kamikaze. Our vision: Destruction of cancer cells by means of exploding bacteria!</p><br />
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<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
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<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><br />
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<p>Documentation of the testing of the components in our genetic circuit.</p><br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
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<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
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</li><br />
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<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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<p style="text-align: center;">Countdown to the <a href="https://2012.igem.org/Regions/Europe/Jamboree" target="_blank">European jamboree</a>:<br />
<div id="jamboreeCountdown"></div><br /><br /><br/><br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_TrondheimTeam:NTNU Trondheim2013-06-16T19:50:44Z<p>Oyas: </p>
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<strong>Judge looking for an overview of what we have done?</strong><br/> We suggest you start off by reading through the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Achievements</a> section. There you will find an overview of the criteria we have fulfilled with links to the relevant documentation. <br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Click to read more about our project, Bacterial Anti-Cancer Kamikaze. Our vision: Destruction of cancer cells by means of exploding bacteria!</p><br />
</div><br />
</div><br />
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<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>Documentation of the testing of the components in our genetic circuit.</p><br />
</div><br />
</div><br />
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<a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
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</li><br />
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<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
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</li><br />
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<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Model"><img alt="" src="https://static.igem.org/mediawiki/2012/d/d0/Model_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach"><img alt="" src="https://static.igem.org/mediawiki/2012/7/70/Outreach_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
</div><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Press"><img alt="" src="https://static.igem.org/mediawiki/2012/2/28/Press_thumb.png"></a><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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<p style="text-align: center;">Countdown to the <a href="https://2012.igem.org/Regions/Europe/Jamboree" target="_blank">European jamboree</a>:<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_TrondheimTeam:NTNU Trondheim2013-06-16T19:49:19Z<p>Oyas: </p>
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<strong>Judge looking for an overview of what we have done?</strong><br/> We suggest you start off by reading through the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Achievements</a> section. There you will find an overview of the criteria we have fulfilled with links to the relevant documentation. <br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Click to read more about our project, Bacterial Anti-Cancer Kamikaze. Our vision: Destruction of cancer cells by means of exploding bacteria!</p><br />
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<a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><br />
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<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
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<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><br />
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<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>Documentation of the testing of the components in our genetic circuit.</p><br />
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<a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><br />
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<p>We have collaborated with the Rose-Hulman Institute of Technology iGEM team.</p><br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Experiments_results_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Collaboration_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
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<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Model"><img alt="" src="https://static.igem.org/mediawiki/2012/d/d0/Model_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
</div><br />
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<ul class="thumbnails"><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach"><img alt="" src="https://static.igem.org/mediawiki/2012/7/70/Outreach_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Press"><img alt="" src="https://static.igem.org/mediawiki/2012/2/28/Press_thumb.png"></a><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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<p style="text-align: center;">Countdown to the <a href="https://2012.igem.org/Regions/Europe/Jamboree" target="_blank">European jamboree</a>:<br />
<div id="jamboreeCountdown"></div><br /><br /><br/><br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-06-16T17:17:00Z<p>Oyas: </p>
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<div class="heading visible-tablet visible-desktop"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
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<span class="sub-heading-small">NTNU IS</span> <span style="font-size: 30pt; font-style: italic;">B.A.C.K.</span><br />
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B<span class="sub-heading-small">acterial</span> A<span class="sub-heading-small">nti</span>-C<span class="sub-heading-small">ancer</span>-K<span class="sub-heading-small">amikaze</span><br />
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<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-bullhorn"></i> Outreach <b class="caret"></b></a><br />
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<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Collaboration</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Human practices</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Press coverage</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><br />
<i class="icon-trophy"></i> Achievements</a><br />
</li><br />
<li class="hidden-navbar"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker" target="_blank"><br />
<i class="icon-heart"></i> Matchmaker</a><br />
</li><br />
</ul><br />
<ul class="nav pull-right hidden-navbar"><br />
<li id="fb-icon"><br />
<a href="http://facebook.com/ntnuigem" target="_blank"><i class="icon-facebook"></i></a><br />
</li><br />
<li id="twitter-icon"><br />
<a href="http://twitter.com/iGEM_NTNU" target="_blank"><i class="icon-twitter"></i></a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-06-16T16:41:54Z<p>Oyas: </p>
<hr />
<div><html lang="en"><br />
</p></div></div></div></div><br />
<head><br />
<meta http-equiv="content-type" content="text/html; charset=utf-8"><br />
<meta charset="utf-8"><br />
<meta name="viewport" content="width=device-width, initial-scale=1.0"><br />
<br />
<!-- Google fonts --> <br />
<link href='http://fonts.googleapis.com/css?family=Ubuntu:400,400italic,700,700italic' rel='stylesheet' type='text/css'><br />
<br />
<!-- Styles --><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/bootstrap.css" rel="stylesheet"><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/jquery.countdown.css" rel="stylesheet"><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/extras.css" rel="stylesheet"><br />
<br />
<!-- Responsive css must be added after top padding --><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/responsive.css" rel="stylesheet"><br />
<br />
<!-- HTML5 shim, for IE6-8 support of HTML5 elements --><br />
<!--[if lt IE 9]><br />
<script src="http://html5shim.googlecode.com/svn/trunk/html5.js"></script><br />
<![endif]--><br />
<!--[if lt IE 7]><br />
<script src="http://ie7-js.googlecode.com/svn/version/2.1(beta4)/IE7.js"></script><br />
<![endif]--><br />
<br />
</head><br />
<br />
<body><br />
<br />
<!-- Facebook box <br />
================================================== --><br />
<div id="fb-root"></div><br />
<script><br />
(function(d, s, id) {<br />
var js, fjs = d.getElementsByTagName(s)[0];<br />
if (d.getElementById(id)) return;<br />
js = d.createElement(s); js.id = id;<br />
js.src = "//connect.facebook.net/en_US/all.js#xfbml=1";<br />
fjs.parentNode.insertBefore(js, fjs);<br />
}(document, 'script', 'facebook-jssdk'));<br />
</script><br />
<br />
<br />
<!-- Header<br />
================================================== --><br />
<div class="header"><br />
<div class="container"><br />
<div class="heading visible-tablet visible-desktop"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
<div class="heading heading-small visible-phone"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
<div class="sub-heading visible-tablet visible-desktop" id="sub-heading1"><br />
<span class="sub-heading-small">NTNU IS</span> <span style="font-size: 30pt; font-style: italic;">B.A.C.K.</span><br />
</div><br />
<div class="sub-heading visible-desktop" id="sub-heading2"><br />
B<span class="sub-heading-small">acterial</span> A<span class="sub-heading-small">nti</span>-C<span class="sub-heading-small">ancer</span>-K<span class="sub-heading-small">amikaze</span><br />
</div><br />
<div class="pull-right visible-desktop" style="text-align:right;" id="ntnu-logo"><br />
<a href="http://ntnu.edu" target="_blank"><img src="https://static.igem.org/mediawiki/2012/3/36/Ntnu_logo_en_scaled.png"/></a><br/><br />
<a href="https://2012.igem.org/Main_Page" target="_blank"><img id="igem-logo" src="https://static.igem.org/mediawiki/2012/c/cc/Igem_logo_scaled.png" /></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!-- Navbar<br />
================================================== --><br />
<div class="navbar navbar-below-header"><br />
<div class="navbar-inner"><br />
<div class="container"><br />
<a class="btn btn-navbar" data-toggle="collapse" data-target=".nav-collapse"><br />
<span class="icon-bar"></span><br />
<span class="icon-bar"></span><br />
<span class="icon-bar"></span><br />
</a><br />
<div class="nav-collapse"><br />
<ul class="nav"><br />
<li><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim"><br />
<i class="icon-home"></i> Home</a><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-info-sign"></i>Project <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Project description</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Experiments and results</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Parts">BioBrick Parts</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Attributions">Attributions</a></li><br />
</ul><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-cogs"></i> Technical stuff <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Notebook">Notebook</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Protocols">Protocols</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-group"></i> Team <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Team">Meet the team</a></li><br />
<li><a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">Official team profile</a></li><br />
</ul><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-bullhorn"></i> Outreach <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Collaboration</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Human practices</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Press coverage</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><br />
<i class="icon-trophy"></i> Achievements</a><br />
</li><br />
<li class="hidden-navbar"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker" target="_blank"><br />
<i class="icon-heart"></i> Matchmaker</a><br />
</li><br />
</ul><br />
<ul class="nav pull-right hidden-navbar"><br />
<li id="fb-icon"><br />
<a href="http://facebook.com/ntnuigem" target="_blank"><i class="icon-facebook"></i></a><br />
</li><br />
<li id="twitter-icon"><br />
<a href="http://twitter.com/iGEM_NTNU" target="_blank"><i class="icon-twitter"></i></a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
</div></div>Oyashttp://2012.igem.org/File:Igem_header_bacteria.pngFile:Igem header bacteria.png2013-06-16T16:14:13Z<p>Oyas: uploaded a new version of &quot;File:Igem header bacteria.png&quot;</p>
<hr />
<div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/FooterTeam:NTNU Trondheim/Templates/Footer2013-06-16T16:11:13Z<p>Oyas: </p>
<hr />
<div><html><br />
</div><!-- /container --><br />
<br />
<br />
<!-- Footer<br />
================================================== --><br />
<br />
<footer class="footer"><br />
<div class="container"><br />
<p class="pull-right top-pointer"><a href="#top"><i class="icon-arrow-up"></i></a></p><br />
<p>Designed and built with <a href="http://www.mediawiki.org/wiki/MediaWiki" target="_blank">MediaWiki</a>, <a href="http://jquery.com/" target="_blank">jQuery</a> and <a href="http://twitter.github.com/bootstrap/" target="_blank">Twitter Bootstrap</a>.</p><br />
<p>Icons by <a href="http://http://fortawesome.github.com/Font-Awesome/" target="_blank">Font Awesome</a>.</p><br />
<p><br /><br />
<i class="icon-envelope-alt"></i><a href="mailto:igem.ntnu@gmail.com"> igem.ntnu@gmail.com</a></p><br />
</div> <!-- /container --><br />
</footer><br />
<br />
<br />
<br />
<!-- Javascript<br />
================================================== --><br />
<!-- Placed at the end of the document so the pages load faster --><br />
<br />
<!-- Bootstrap --><br />
<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.7.2/jquery.min.js"></script><br />
<script src="http://oveoyas.github.io/igem-ntnu-2012/js/bootstrap.min.js"></script><br />
<br />
<script type="text/javascript"><br />
$('.dropdown-toggle').dropdown()<br />
$('.carousel').carousel()<br />
</script><br />
<br />
<!-- Smooth scroll to top --><br />
<script type="text/javascript"><br />
$("a[href='#top']").click(function() {<br />
$("html, body").animate({ scrollTop: 0 }, "medium");<br />
return false;<br />
});<br />
</script><br />
<br />
<!-- jQuery countdown --><br />
<script type="text/javascript" src="http://oveoyas.github.io/igem-ntnu-2012/js/jquery.countdown.js"></script><br />
<script type="text/javascript"><br />
$(function () {<br />
var jamboreeDay = new Date();<br />
jamboreeDay = new Date(2012, 10 - 1, 5);<br />
$('#jamboreeCountdown').countdown({<br />
until: jamboreeDay,<br />
/*onExpiry: liftOff*/<br />
compact: false,<br />
<br />
});<br />
});<br />
/*function liftOff() { <br />
alert('We are rolling!'); <br />
}*/<br />
</script><br />
<br />
<!-- Navigation scroll follow --><br />
<script type="text/javascript"><br />
$(window).scroll(function () { <br />
var scrollPos = $(window).scrollTop();<br />
if (scrollPos > 150) {<br />
$(".navbar").addClass("navbar-fixed-top");<br />
$(".navbar").removeClass("navbar-below-header");<br />
} else {<br />
$(".navbar").removeClass("navbar-fixed-top");<br />
$(".navbar").addClass("navbar-below-header");<br />
}<br />
if (scrollPos > 180) {<br />
$(".toc").addClass("stickBelowNavigation");<br />
} else {<br />
$(".toc").removeClass("stickBelowNavigation");<br />
}<br />
});<br />
</script><br />
<br />
<!-- iGem wiki hacks --><br />
<br />
<!-- Remove all empty <p> tags --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
$("p").filter( function() {<br />
return $.trim($(this).html()) == '';<br />
}).remove();<br />
});<br />
</script><br />
<br />
<!-- Remove "team" from the menubar --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
$("menubar > ul > li:last-child").remove();<br />
});<br />
</script><br />
<br />
<!-- Empty heading? - Then remove it.. --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
if ("" == "Vision") {<br />
$("#heading").remove();<br />
}<br />
});<br />
</script><br />
<br />
<!-- Fill the screen of index page on tablets --><br />
<script type="text/javascript"><br />
$(document).ready(function() {f<br />
if ( $(window).width() < 1100 ) {<br />
$('#index-collapse').addClass('span12').removeClass('span9');<br />
}<br />
else {<br />
$('#index-collapse').removeClass('span12').addClass('span9');<br />
}<br />
});<br />
</script><br />
<br />
</body><br />
</html></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-06-16T16:09:47Z<p>Oyas: </p>
<hr />
<div><html lang="en"><br />
</p></div></div></div></div><br />
<head><br />
<meta http-equiv="content-type" content="text/html; charset=utf-8"><br />
<meta charset="utf-8"><br />
<meta name="viewport" content="width=device-width, initial-scale=1.0"><br />
<br />
<!-- Google fonts --> <br />
<link href='http://fonts.googleapis.com/css?family=Ubuntu:400,400italic,700,700italic' rel='stylesheet' type='text/css'><br />
<br />
<!-- Styles --><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/bootstrap.css" rel="stylesheet"><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/jquery.countdown.css" rel="stylesheet"><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/extras.css" rel="stylesheet"><br />
<br />
<!-- Responsive css must be added after top padding --><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/responsive.css" rel="stylesheet"><br />
<br />
<!-- HTML5 shim, for IE6-8 support of HTML5 elements --><br />
<!--[if lt IE 9]><br />
<script src="http://html5shim.googlecode.com/svn/trunk/html5.js"></script><br />
<![endif]--><br />
<!--[if lt IE 7]><br />
<script src="http://ie7-js.googlecode.com/svn/version/2.1(beta4)/IE7.js"></script><br />
<![endif]--><br />
<br />
</head><br />
<br />
<body><br />
<br />
<!-- Facebook box <br />
================================================== --><br />
<div id="fb-root"></div><br />
<script><br />
(function(d, s, id) {<br />
var js, fjs = d.getElementsByTagName(s)[0];<br />
if (d.getElementById(id)) return;<br />
js = d.createElement(s); js.id = id;<br />
js.src = "//connect.facebook.net/en_US/all.js#xfbml=1";<br />
fjs.parentNode.insertBefore(js, fjs);<br />
}(document, 'script', 'facebook-jssdk'));<br />
</script><br />
<br />
<br />
<!-- Header<br />
================================================== --><br />
<div class="header"><br />
<div class="container"><br />
<div class="heading visible-tablet visible-desktop"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
<div class="heading heading-small visible-phone"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
<div class="sub-heading visible-tablet visible-desktop" id="sub-heading1"><br />
<span class="sub-heading-small">NTNU IS</span> <span style="font-size: 30pt; font-style: italic;">B.A.C.K.</span><br />
</div><br />
<div class="sub-heading visible-desktop" id="sub-heading2"><br />
B<span class="sub-heading-small">acterial</span> A<span class="sub-heading-small">nti</span>-C<span class="sub-heading-small">ancer</span>-K<span class="sub-heading-small">amikaze</span><br />
</div><br />
<div class="pull-right visible-desktop" style="text-align:right;" id="ntnu-logo"><br />
<a href="http://ntnu.edu" target="_blank"><img src="https://static.igem.org/mediawiki/2012/3/36/Ntnu_logo_en_scaled.png"/></a><br/><br />
<a href="https://2012.igem.org/Main_Page" target="_blank"><img id="igem-logo" src="https://static.igem.org/mediawiki/2012/c/cc/Igem_logo_scaled.png" /></a><br />
</div><br />
</div><br />
</div><br />
<br />
<!-- Navbar<br />
================================================== --><br />
<div class="navbar navbar-below-header"><br />
<div class="navbar-inner"><br />
<div class="container"><br />
<a class="btn btn-navbar" data-toggle="collapse" data-target=".nav-collapse"><br />
<span class="icon-bar"></span><br />
<span class="icon-bar"></span><br />
<span class="icon-bar"></span><br />
</a><br />
<div class="nav-collapse"><br />
<ul class="nav"><br />
<li class="active"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim"><br />
<i class="icon-home"></i> Home</a><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-info-sign"></i>Project <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Project description</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Modelling</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Experiments and results</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Parts">BioBrick Parts</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Attributions">Attributions</a></li><br />
</ul><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-cogs"></i> Technical stuff <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Notebook">Notebook</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Protocols">Protocols</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Safety">Safety</a></li><br />
</ul><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-group"></i> Team <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Team">Meet the team</a></li><br />
<li><a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">Official team profile</a></li><br />
</ul><br />
</li><br />
<li class="dropdown"><br />
<a href="#" class="dropdown-toggle" data-toggle="dropdown"><i class="icon-bullhorn"></i> Outreach <b class="caret"></b></a><br />
<ul class="dropdown-menu"><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Collaboration</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Human practices</a></li><br />
<li><a href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Press coverage</a></li><br />
</ul><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><br />
<i class="icon-trophy"></i> Achievements</a><br />
</li><br />
<li class="hidden-navbar"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker" target="_blank"><br />
<i class="icon-heart"></i> Matchmaker</a><br />
</li><br />
</ul><br />
<ul class="nav pull-right hidden-navbar"><br />
<li id="fb-icon"><br />
<a href="http://facebook.com/ntnuigem" target="_blank"><i class="icon-facebook"></i></a><br />
</li><br />
<li id="twitter-icon"><br />
<a href="http://twitter.com/iGEM_NTNU" target="_blank"><i class="icon-twitter"></i></a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</div><br />
</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2013-06-16T16:04:28Z<p>Oyas: </p>
<hr />
<div><html lang="en"><br />
</p></div></div></div></div><br />
<head><br />
<meta http-equiv="content-type" content="text/html; charset=utf-8"><br />
<meta charset="utf-8"><br />
<meta name="viewport" content="width=device-width, initial-scale=1.0"><br />
<br />
<!-- Google fonts --> <br />
<link href='http://fonts.googleapis.com/css?family=Ubuntu:400,400italic,700,700italic' rel='stylesheet' type='text/css'><br />
<br />
<!-- Styles --><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/bootstrap.css" rel="stylesheet"><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/jquery.countdown.css" rel="stylesheet"><br />
<link href="http://oveoyas.github.io/igem-ntnu-2012/css/extras.css" rel="stylesheet"><br />
<br />
<!-- Responsive css must be added after top padding --><br />
<link href="http://folk.ntnu.no/oyas/igem3/bootstrap2/css/responsive.css" rel="stylesheet"><br />
<br />
<!-- HTML5 shim, for IE6-8 support of HTML5 elements --><br />
<!--[if lt IE 9]><br />
<script src="http://html5shim.googlecode.com/svn/trunk/html5.js"></script><br />
<![endif]--><br />
<!--[if lt IE 7]><br />
<script src="http://ie7-js.googlecode.com/svn/version/2.1(beta4)/IE7.js"></script><br />
<![endif]--><br />
<br />
</head><br />
<br />
<body><br />
<br />
<!-- Facebook box <br />
================================================== --><br />
<div id="fb-root"></div><br />
<script><br />
(function(d, s, id) {<br />
var js, fjs = d.getElementsByTagName(s)[0];<br />
if (d.getElementById(id)) return;<br />
js = d.createElement(s); js.id = id;<br />
js.src = "//connect.facebook.net/en_US/all.js#xfbml=1";<br />
fjs.parentNode.insertBefore(js, fjs);<br />
}(document, 'script', 'facebook-jssdk'));<br />
</script><br />
<br />
<br />
<!-- Header<br />
================================================== --><br />
<div class="header"><br />
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</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T23:37:05Z<p>Oyas: /* Colicin (BBa_K822002) */</p>
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<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
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__TOC__<br />
==Introduction==<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
==Colicin (<partinfo>BBa_K822002</partinfo>)==<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{| style="margin: 1em auto 1em auto; width: auto;"<br />
|style="vertical-align: top;"|[[File:Colisin2.png|thumb|center|450px|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)]]<br />
|style="vertical-align: top;"|[[File:Colisin_1.png|thumb|center|450px|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)]]<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added.<br />
<br />
==YFP generator (<partinfo>BBa_K822003</partinfo>)==<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
==Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)==<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
{|border="0" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|thumb|center|300px|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.]]<br />
|style="vertical-align: top;"|[[File:RBS+LacI+term-gel.PNG|thumb|center|450px|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. ]]<br />
|}<br />
<br />
==lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)==<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
==ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)==<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T23:36:31Z<p>Oyas: /* Colicin (BBa_K822002) */</p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
==Introduction==<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
==Colicin (<partinfo>BBa_K822002</partinfo>)==<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{| style="margin: 1em auto 1em auto; width: auto;"<br />
|style="vertical-align: top;"|[[File:Colisin2.png|thumb|center|450px|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)]]<br />
|[[File:Colisin_1.png|thumb|center|450px|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)]]<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added.<br />
<br />
==YFP generator (<partinfo>BBa_K822003</partinfo>)==<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
==Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)==<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
{|border="0" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|thumb|center|300px|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.]]<br />
|style="vertical-align: top;"|[[File:RBS+LacI+term-gel.PNG|thumb|center|450px|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. ]]<br />
|}<br />
<br />
==lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)==<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
==ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)==<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
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{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/TeamTeam:NTNU Trondheim/Team2012-09-26T23:30:18Z<p>Oyas: </p>
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<h1>Meet the team <small> Team NTNU Trondheim up close and personal</small></h1><br />
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<h1>The students</h1><br />
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<dl><div class="well well-small"><br />
<dt>Origin of replication:</dt><dd>Trondheim</dd><br />
<dt>Study program:</dt><dd>Graduated M.Sc. in Chemical engineering and biotechnology from NTNU, with specialization in biotechnology. <!--So now, I'm a graduate engineer (yay!). In my master thesis, I investigated the interaction between DNA and Uracil DNA Glycosylase, which is a repair enzyme removing uracil from DNA, using optical tweezers. I also just recieved a research stipend in medical imaging, meaning that for the next semester, I'll continue investigating single molecule interactions using optical tweezers.--></dd></div><br />
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<h2>Gunvor Røkke</h2><br />
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<dt>Interests outside academia:</dt><dd>Mainly playing the violin. I have been playing since I was five years old, and I am currently leading one of the three folk music orchestras in Trondheim. I also like to swim, or to gab with my friends.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul> <li><i>Team leader:</i> Teaching the other team members the basic cloning techniques used for biobrick assembly.</li><br />
<li><i>Team representative:</i> In case of interested journalists, Gunvor will talk to them. Rolf will be her manager and Nina her stylist! </li><br />
<li><i>Team dietitian:</i> One of Gunvor's less serious responsibility areas is to make sure that all team members get their daily dose of carbohydrates (as we all agree that lowcarb is nonsense).</li><br />
<li><i>Vice PR chief: </i>Rolf is the actual PR chief, but in case Rolf meets some really stubborn journalists, and he gives up, Gunvor will take over.</li></dd><br />
<dt>Fun fact:</dt><dd>I'm able to whistle and hum in two part harmony with myself. And no, I'm not mutated.</dd><br />
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<dt>Origin of replication:</dt><dd>Eidsvåg, Møre og Romsdal</dd><br />
<dt>Study program:</dt><dd>M.Sc. in Chemical engineering and biotechnology at NTNU, with specialization in biotechnology.</dd></div><br />
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<h2>Nina Hole</h2><br />
<dl><br />
<dt>Interests outside academia:</dt><dd>Sports enthusiast, but especially interested in football. I also play football myself on NTNU's own football team. Other interests are reading, traveling and food.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul><li><i>Gunvor's stylist</i>:If Gunvor is representing the team, I have the main responsibility to do her make-up and so on.</li><li><i>Photo chief</i>: If something needs photographing, Nina is on the job!</li><br />
<li><i>Facebook and twitter chief: </i>Updating facebook and twitter is Nina's responsibility.</li><br />
<li><i>Travel chief: </i>Nina is travel chief, and have had the main responsibility for booking our hotel in Amsterdam. Apparently, she's good at this, because the hotell looks absolutely fabulous!</li><br />
<li><i>Megan Fox chief: </i>Internal humour. Don't ask...</li></ul><br />
</dd><br />
<dt>Fun fact:</dt><dd><br />
My favorite author/musician Jo Nesbø, has named his main character in his criminal novels Harry Hole. His inspiration for the last name comes from my family! </dd><br />
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<dt>Origin of replication:</dt><dd>Drammen</dd><br />
<dt>Study program:</dt><dd>M.Sc. in Chemical engineering and biotechnology at NTNU, with specialization in biotechnology.</dd></div><br />
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<h2>Jarle Pahr</h2><br />
<dl><br />
<dt>Interests outside academia:</dt><dd>Aikido and psychology.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul><li><i>Master of the notebook:</i> Trying to get the other team members to update the notebook.</li><li><i>Cake chief: </i> Baking awesome cakes.</li><br />
<li><i>Plate chief: </i>Keeping track of all the agar plates in the fridge is Jarle's job.</li><br />
<li><i>Culture chief: </i>Keeping track of cell cultures in the fridge, in the incubator, or on glycerol stock in the freezer.</ul></dd><br />
<dt>Fun fact:</dt><dd>Is resistant to norovirus, thanks to a mutation in the FUT2 gene. Being a mutant is nice!</dd><br />
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<dt>Origin of replication:</dt><dd>Larvik</dd><br />
<dt>Study program:</dt><dd>M.Sc. in Chemical engineering and biotechnology at NTNU, with specialization in biotechnology.</dd></div><br />
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<h2>Eirin Korvald</h2><br />
<dl><br />
<dt>Interests outside academia:</dt><dd>Music (both playing and listening), snowboarding, hiking, knitting, hanging with friends.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul> <li><i>iGEM hair dresser:</i> Making sure the entire team has fabulous hair at all times.</li><br />
<li><i>Cake chief: </i>Eirin is keeping track of the cakes the different team members owe the rest of the team.</li><br />
<li><i>Supervisor: </i>It's also Eirin's job to keep an eye on the rest of the team in Amsterdam, to make sure we don't get lost there.</li></dd><br />
<dt>Fun fact:</dt><dd>Is able to cut her own hair - nicely!.</dd><br />
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<dt>Origin of replication:</dt><dd>Trondheim</dd><br />
<dt>Study program:</dt><dd>M.Sc. in Chemical engineering and biotechnology at NTNU, with specialization in biotechnology.</dd></div><br />
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<h2>Ove Øyås</h2><br />
<dl><br />
<dt>Interests outside academia:</dt><dd>Nature, cycling, hiking, music, programming, being enthusiastic about stuff.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul> <li><i>Internet chief:</i> Fixing the wiki and the Matchmaker. Making spaghetti code.</li><br />
<li><i>T-shirt chief: </i>Has been in charge of ordering team t-shirts. The rest of us haven't seen them yet, but we have high expectations!</li><br />
<li><i>Vice facebook and twitter chief: </i>Is updating facebook and twitter together with Nina.</li></dd><br />
<dt>Fun fact:</dt><dd>Will easily eat raw ginger and garlic.</dd><br />
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<dt>Origin of replication:</dt><dd>Trondheim</dd><br />
<dt>Study program:</dt><dd>M.Sc. in Chemical engineering and biotechnology at NTNU, with specialization in applied theoretical chemistry.</dd></div><br />
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<h2>Rolf Heilemann Myhre</h2><br />
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<dt>Interests outside Academia:</dt><dd>In my spare time, I like exercising, hanging with friends, partying, movies etc. Also, I can't live without my computer.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul>Modelling the genetic circuit.<br />
<li><i>PR chief: </i>Will be talking to a (hopefully) enormous crowd of journalists.</li><br />
<li><i>Manager for Gunvor: </i>It's Rolf responsibility to make sure Gunvor says the right things to the journalists.</li><br />
<li><i>Cleaning chief: </i>Is making sure the NTNU iGEM headquarters looks tidy.</li></dd><br />
<dt>Fun fact:</dt><dd>I have not studied any biotechnology before joining the iGEM team.</dd><br />
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<h1>The advisors</h1><br />
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<dt>Origin of replication:</dt><dd>Stavanger, Norway</dd><br />
<dt>Position:</dt><dd>Professor</dd><br />
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<h2>Eivind Almaas</h2><br />
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<dt>Area of expertise:</dt><dd>Systems biology and network analysis</dd><br />
<dt>Interests outside academia:</dt><dd>Hiking, reading, living.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul> <li>Team organization.</li><li>Computer modelling.</li></dd><br />
<dt>Fun fact:</dt><dd>Big fan of 50's Rock'n Roll and Rockabilly.</dd><br />
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<dt>Origin of replication:</dt><dd>Cologne, Germany</dd><br />
<dt>Position:</dt><dd>Postdoctoral fellow</dd><br />
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<h2>Rahmi Lale</h2><br />
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<dt>Area of expertise:</dt><dd>Transcriptional and translational regulation of bacterial gene<br />
expression, metabolic engineering. Metagenome/biodiscovery.<br />
</dd><br />
<dt>Interests outside academia:</dt><dd> Loves playing bass, likes biking/hiking/skiing/fishing/sailing, into web/graphic design.</dd><br />
<dt>Areas of responsibility:</dt><dd>Herding the nerds!</dd><br />
<li><i>Social chief: </i>Rahmi was chosen to be social chief at the beginning of the summer. It's his responsibility to make sure the team is also doing social stuff not related to lab work.</li><br />
<dt>Fun fact:</dt><dd>He loves bugs so much that he makes his own kefir.</dd><br />
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<dt>Origin of replication:</dt><dd>Mannheim, Germany</dd><br />
<dt>Position:</dt><dd>Associate professor</dd></div><br />
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<h2>Martin Hohmann-Marriott</h2><br />
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<dt>Area of expertise:</dt><dd>Photosynthesis, bioenergetics, molecular biology.</dd><br />
<dt>Interests outside academia:</dt><dd>My family, history, all things computer and technology</dd><br />
<dt>Areas of responsibility:</dt><dd>Instructor, molecular biology and physiology<br />
</dd><br />
<dt>Fun fact:</dt><dd>Martin designed exercise equipment for ants as an undergraduate.</dd><br />
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<dt>Origin of replication:</dt><dd>Kristiansand, Norway</dd><br />
<dt>Position:</dt><dd>Doctoral student. Graduated M.Sc. in Physics and mathematics from NTNU.</dd><br />
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<h2>Marius Eidsaa</h2><br />
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<dt>Area of expertise:</dt><dd>Systems biology, mainly focusing on networks, both generally and in biology. I'm currently working on network methods and analysis of microarray data..</dd><br />
<dt>Interests outside academia:</dt><dd>I like to sing, and I'm currently singing tenor in two student-society based choirs. Other interests include music in general, food and drink and good company.</dd><br />
<dt>Areas of responsibility:</dt><dd><ul> <li>I'm a modelling instructor, so my main responsibility is to help set up and analyze our model.</li><br />
<li><i>Barbeque chief: </i>Marius is barbecue chief, and is supposed to be organizing barbeque everytime the weather in Trondheim is nice, which is, unfortunately, not that often.</dd><br />
<dt>Fun fact:</dt><dd>Has been swimming at a national level.</dd><br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/CollaborationTeam:NTNU Trondheim/Collaboration2012-09-26T23:28:17Z<p>Oyas: /* RHIT collaboration */</p>
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<h1>Collaboration <small>Bringing iGEM teams together</small></h1><br />
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==RHIT collaboration==<br />
<br />
During the summer we have had close contact with the Rose-Hulman team from Terre Haute, Indiana. Our main advisor, Dr. Eivind Almaas, was present at another event at the Rose-Hulman university and came in contact with the team advisor from Rose-Hulman, Dr. Richard Anthony. They began discussing the possibility of a collaboration between the two teams, and 7/23/2012 we had our first video conference so the team members could meet and discuss what we could help each other with.<br />
<br />
[[File:Ntnu_rhit_collaboration.png|thumb|center|800px|Our advisor, professor Eivind Almaas, with associate professor Richard Anthony, advisor of the RHIT iGEM team.]]<br />
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We agreed that the Rose-Hulman team would help us characterize our new pLLD BioBrick, and we would in return critique their mathematical model, and make a stochastic model of their system. We had a new video conference in august to update each other, and share experiences about the iGEM project as both teams are fairly new in this competition. This has been a fun experience, and exiting that we can help each other and share experiences across the globe!<br />
<br />
We hope to meet the Rose-Hulman team in Boston in November, and to further continue our cooperation. The work we did for RHIT is available on our [https://2012.igem.org/Team:NTNU_Trondheim/Model#Modelling_for_RHIT modelling page].<br />
<br />
==The iGEM Matchmaker==<br />
<br />
In the middle of the summer, we got the idea of the [https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker Matchmaker]. What brought us to this idea was the fact that if you haven't already arranged a cooperation with another iGEM team, it is hard to find someone to cooperate with. We also knew that the NTNU iGEM team 2011 made many attempts to cooperate with other teams by sending out several emails, but they never recieved any answers.<br />
So we decided to make the Matchmaker, and so far, we think it has been very useful. Hopefully, you think so too! Even if it came quite late in the summer, it was used by several teams, and we hope it can be used by future iGEM teams as well.<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/ModelTeam:NTNU Trondheim/Model2012-09-26T23:26:39Z<p>Oyas: /* Modelling for RHIT */</p>
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<h1>Modelling <small>Stochastic simulations of the genetic circuit</small></h1><br />
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==Overview==<br />
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To get a better understanding of the dynamics of the system, we made a model of the system using the Cain software[http://cain.sourceforge.net] for stochastic simulations. While stochastic simulations are more computationally demanding than deterministic models based on solving ODE's, they allow for random fluctuations that may have a big impact on the system in a cell[http://dx.doi.org/10.1038/nrg1615].<br />
<br />
As we want our system to react to two different signals, three promoters were required. One to respond to lactate, one to respond to low oxygen and a third to respond to signal molecules controlled by the two other promoters. The last promoter would then control cell lysis. For lactate sensing, we have adapted the lld promoter of ''E. coli'', the vgb promoter from ''Vitreoscilla'' is used for the oxygen while the lux promoter from ''Vibrio fischeri'' was used for lysis control.<br />
<br />
The model can be divided into three parts; the lld promoter, the vgb promoter and the lux promoter. Each of these systems was first modelled separately. This made it easier to observe the effect of changing individual parameters and make reasonable estimates when experimental data were not available. The final model contained 66 reactions and 46 parameters, many of which were estimates. However, we experienced that working with the model and observing how parameter changes influenced protein expressions gave valuable insight into the system.<br />
<br />
Stochastic models, as opposed to deterministic models, describe the presence of species with the number of molecules. For small molecules, in this case oxygen and lactate, this gives very high numbers. For example; a 1 mmol concentration of lactate gives about 400000 molecules in the cell assuming a cell volume of 0.7 µm³. This many molecules are computationally demanding to keep track of and the diffusion of these molecules through the cell membrane is relatively fast[http://dx.doi.org/10.1002/bit.260320308]. To simplify, lower, constant concentrations and higher propensity functions were used. A possible problem with this simplification is greater fluctuations than a more realistic model.<br />
<br />
In general, the models seems to be too sensitive with small stimulations giving very large effects. A possible reason is that the values we used for the translation rates are too high, as these are estimates. Another possibility is that the propensity for mRNA decay are more different between deterministic models, as in the references, and stochastic models than expected. This was especially the case for the lux promoter.<br />
<br />
All the equations in the model assumed mass action. For the details and parameters used in the model, see [[Team:NTNU_Trondheim/Equations|Equations]].<br />
<br />
A .zip file of the full model can be downloaded [[Media:NTNU_Trondheim_Full.zip|here]].<br />
<br />
==Lld promoter==<br />
In ''E. coli'', the Lld promoter regulates the metabolism of l-Lactate[http://dx.doi.org/10.1128/JB.02013-07]. Two proteins involved in metabolism as well as the regulatory protein LldR is induced by the presence of lactate. LldR in solution will form dimers that will bind to two sites on the promoter DNA. This will block RNA polymerase from binding to the DNA, possibly with a looping structure[http://dx.doi.org/10.1128/JB.02013-07 [4<nowiki>]</nowiki>]. If l-Lactate is present in sufficient quantities, it will bind to the LldR dimer and cause a reconformation of the complex. This activates the promoter. A similar system is found in Corynebacterium Glutamicum[http://dx.doi.org/10.1128/JB.01147-07]. <br />
<br />
We isolated the promoter part of the operon and coupled it to expression of the LuxI protein from V. Ficheri[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0061]. As LldR is already present in ''E. Coli'', it was assumed to have an initial concentration as well as a constant production and degradation. A more realistic system could have included a self regulating feedback system, but this would further complicate the model without necessarily increasing the descriptive accuracy.<br />
<br />
Binding to the promoter was modelled as LldR in solution forming dimers that could bind to the promoter. Lactate would then bind to the dimer, either in solution or bound to DNA. This gave a quite reasonable behaviour in the presence or absence of lactate, but other paths are also possible. We were not able to find any detailed description of the mechanism.<br />
<br />
The plots show the production of the LuxI protein as a response to the presence of lactate. The plot on the left shows a system with a quite low concentration of lactate, 10 in the model. Under these conditions, only a few, less than 5, promoters are activated and, as result, the LuxI concentration remains low.The plot on the right shows the response to a 100 times larger lactate concentration. Even though the number of active promoters remains relatively low, the constant production of mRNA leads to a large production of LuxI.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_LldrLow.png|thumb|center|450px|Figure 1. Response of lld promoter to a lactate level of 10]]<br />
|[[File:NTNU_Trondheim_Model_LldrHigh.png|thumb|center|450px|Figure 2. Response of lld promoter to a lactate level of 1000]]<br />
|}<br />
<br />
==Vgb promoter==<br />
''Vitreoscilla'' is a gram-negative, aerobic bacteria[http://dx.doi.org/10.1016/j.micres.2005.03.004]. To facilitate continued oxygen uptake, it produces bacterial hemoglobin in low oxygen environments. This production is regulated by the vgb promoter[http://partsregistry.org/wiki/index.php?title=Part:BBa_K561001] that has little or low expression in aerobic and anaerobic conditions, but is strongly expressed in microaerobic conditions[http://jb.asm.org/content/171/11/5995]. This behaviour is caused by two proteins; Fnr and ArcA[http://dx.doi.org/10.1016/j.micres.2005.03.004 [7<nowiki>]</nowiki>]. <br />
<br />
Both of these proteins are also found in ''E. Coli'' and are involved in the regulation of at least 20 different processes in the cell[http://dx.doi.org/10.1111/j.1574-6968.1990.tb04109.x]. Fnr contains an [2Fe-2S]²⁺ cluster that is directly oxidized in the precence of oxygen[http://dx.doi.org/10.1016/S0014-5793(97)01219-2]. This was modelled as the unoxidized protein binding to the promoter, thus activating it. In the precense of high oxygen concentrations, however, it would be converted to an oxidized form that did not bind to the promoter.<br />
<br />
For the ArcA protein, the system is more complicated. In anaerobic conditions, a membrane protein called ArcB will autophosphorylate. This will in turn phosphorylate ArcA which can then inhibit the vgb promoter[http://dx.doi.org/10.1126/science.1059361]. To simplify the model, this was simulated the same way as the Fnr protein with direct oxidation, but with a higher oxidation propensity and the binding of the reduced protein caused inhibition, not activation. This gave a qualitative description of the system with little expression with high or very low oxygen concentrations and activation at intermediate levels.<br />
<br />
In the figures below, the LuxR production and amount of active promoter is plotted for three different oxygen levels; 10, 250 and 2000. For the levels with low induction, the amount of protein remains relatively low around 100. In the high induction case, however, the concentration rapidly increases to over 900. Even for the high induction case, the amount of activated promoter remains relatively low.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_VgbLow.png|thumb|center|450px|Figure 3. Response of vgb promoter to an oxygen level of 10]]<br />
|[[File:NTNU_Trondheim_Model_VgbHigh.png|thumb|center|450px|Figure 4. Response of vgb promoter to an oxygen level of 250]]<br />
|}<br />
[[File:NTNU_Trondheim_Model_VgbVeryHigh.png|thumb|center|450px|Figure 5. Response of vgb promoter to an oxygen level of 1000]]<br />
<br />
==Lux promoter==<br />
To control the production of holin, which causes cell lysis, see full model, the lux promoter from ''Vibrio fischeri'' were used. V. fischeri lives in the light emitting organs of certain marine animals. When floating freely in the ocean, the bacteria do not produce light, however, in the light organs, the bacteria will achive high concentrations and start to luminesce[http://dx.doi.org/10.1146/annurev.micro.50.1.727]. Luminescense is controlled by the lux promoter that is activated by a complex of the protein LuxR and homoserine lactone (HSL). <br />
<br />
HSL is a relatively small signalling molecule that is formed by the reaction between S-adenosylmethionine (SAM) and 3-oxo-hexanoyl-CoA (hex). This reaction is catalyzed bu the LuxI enzyme. LuxI is constitutively expressed in the cells, but HSL will diffuse through the cell membrane and not reach high enough concentrations to activate the promoter if there is no HSL in the environment surrounding the cell. At high cell concentrations the amount of HSL, however, a buildup of HSL will activate the promoter. The lux promoter has been adapted as a biobrick and used successfully in ''E. coli''[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062].<br />
<br />
To model the production of HSL, a constant production and degradation of SAM and hex was assumed. In the presence of LuxI a complex would form and HSL would be produced, consuming the substrates. The diffusion of HSL was modelled as a first order mass action equation with HSL going to the null space.<br />
<br />
In the cell, two LuxR proteins will form a complex with two HSL molecules. This complex will then bind to a palindromic site and activate the promoter. In the model, the reaction was described as two HSL and two proteins reacting directly and forming a complex that activated the promoter. The results are shown in the figure below. In the figure to the left, a constant production of 0.001 s⁻¹ was assumed for both LuxR and LuxI. This gave an expression of Holin, the compound that cause lysis, see full model, with a maximum about 700. This was quite high, but less than the lethal amount of about a 1000[http://dx.doi.org/10.1146/annurev.micro.54.1.799]. After increasing the expression to 0.01 s⁻¹, the amount increased to about 5500. Again, the amount of activated promoter were quite low, indicating a too high transcription and translation rate.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_LuxBoxLow.png|thumb|center|450px|Figure 6. Response of lux promoter to a LuxI and LuxR production rate of 0.001 s⁻¹]]<br />
|[[File:NTNU_Trondheim_Model_LuxBoxHigh.png|thumb|center|450px|Figure 7. Response of lux promoter to a LuxI and LuxR production rate of 0.01 s⁻¹]]<br />
|}<br />
<br />
==Full model==<br />
In the full model, the previous parts were put together and made to control the lysis genes. The lysis genes[http://partsregistry.org/wiki/index.php/Part:BBa_K112808] are adapted from the T4 phage. T4 infect gram negative bacteria and cause lysis in them. This is caused by two compounds; a membrane protein called holin and an endolysin that degrades cell walls. The endolysin itself is relatively harmless for the bacteria, as it cannot pass through the inner membrane[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>]. To facilitate lysis, holin is required as it creates pores in the inner membrane. About 1000 holin molecules per cell is required for lysis[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>].<br />
<br />
For the phage, there is an optimal delay between infection and lysis, as it takes time to assemble viruses in the cell. To delay lysis, the genes for an antiholin is also present in the gene cassette. Antiholin forms a complex with holin, thus disabling the creation of pores. An infected bacteria will behave normally up until lysis begins[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>].<br />
<br />
The figures below describe the behaviour of the system under various conditions. Figure 8 shows the system with a relatively low oxygen level of 100 and a high lactate level of 1000. The antiholin will cause a delay in lysis, but holin will reach lethal levels in about an hour. Figure 9 shows the response to oxygen levels of 2000 and lactate levels of 10. In this case, there is still holin production, but most of it is complexed with antiholin and it does not reach lethal levels.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_HolinHolinHigh.png|thumb|center|450px|Figure 8. Production rate of holin at oxygen levels of 100 and lactate levels of 1000]]<br />
|[[File:NTNU_Trondheim_Model_HolinHolinLow.png|thumb|center|450px|Figure 9. Production rate of holin at oxygen levels of 2000 and lactate levels of 10]]<br />
|}<br />
<br />
Figure 10 shows the production of LuxI and LuxR with maximally inducing oxygen levels, about 250, but no lactate. There is much LuxR being produced, but no LuxI and thus no HSL. In the model, it turned out to be difficult to turn the system completely off, as long as there were lactate present, even in small amounts. This is probably not realistic, as V. fischeri has a constant production of both LuxR and LuxI without inducing luminescence. <br />
<br />
The last plot shows the response to high oxygen levels; 5000, and high lactate levels; 100. this gives a response similiar to the one in Figure 9 were holin is produced, but forms a complex with antiholin and does not reach large enough quantities to cause lysis.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_HighOxNoLact.png|thumb|center|450px|Figure 10. Production rate of LuxR and LuxI at oxygen levels of 250 and no lactate]]<br />
|[[File:NTNU_Trondheim_Model_LactVHighOx.png|thumb|center|450px|Figure 11. Production rate of holin at oxygen levels of 5000 and lactate levels of 100]]<br />
|}<br />
<br />
==Modelling for RHIT==<br />
<br />
Our part of the [https://2012.igem.org/Team:NTNU_Trondheim/Collaboration collaboration] with RHiT was helping them with stochastic modelling of the trigger system for mating in yeast. The full mechanism has been quite well studied, but it is very complicated[http://dx.doi.org/10.1002/yea.1122]. In RHiTs [[Team:RHIT/Modeling|model]], the final steps of the mechanism is activation of the Ste12 protein by the Fus3 enzyme. To simplify the model, production of Fus3 in the model was described by a sigmoid curve found in experiments[http://dx.doi.org/10.1038/nature08946] with respect to the concentration of &alpha;-pheromone. Inactive Ste12 was quickly activated by the presence of Fus3, so the outcome of active Ste12 followed a similar sigmoid curve, giving the expected switch behaviour. The resulting plot is shown in Figure 1. Each point is the average of 100 trajectories with the error bars indicating one standard deviation.<br />
<br />
[[File:NTNU_Trondheim_YeastAlpha.png|thumb|center|450px|Figure 1. Amount of activated Ste12 at steady state as a response to &alpha;-pheromone concentrations. Error bars show one standard deviation.]]<br />
<br />
The equations used in the model are given in the table below. The parameters are taken from[http://dx.doi.org/10.1002/yea.1122 [1<nowiki>]</nowiki>] and are modelled using mass action solvers, except Fus3 &rarr; Fus3PP, which use a sigmoid function. Timesteps in the model are minutes.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Reaction<br />
!Propensity<br />
!Comment<br />
|-<br />
|Fus3 &rarr; Fus3PP<br />
|*<br />
|Activation of Fus3<br />
|-<br />
|Fus3PP &rarr; Fus3<br />
|150<br />
|Deactivation of Fus3<br />
|-<br />
|Fus3PP + Ste12 &rarr; Fus3Ste12<br />
|18<br />
|Activation of Ste12 through complexation with Fus3<br />
|-<br />
|Fus3Ste12 &rarr; Fus3PP + Ste12<br />
|10<br />
|Deactivation of Ste12 by release of Fus3<br />
|-<br />
|Bar1 + Fus3Ste12 &rarr; aBar1 + Fus3Ste12<br />
|0.1<br />
|Activation of Bar1 enzyme<br />
|-<br />
|aBar1 &rarr; Bar1<br />
|0.1<br />
|Deactivation of Bar1<br />
|-<br />
|aBar1 &rarr; ø<br />
|0.01<br />
|Export of active Bar1<br />
|}<br />
''* The function for Fus3 activation is given by'' 200*&alpha;⁶/(&alpha;⁶ + 150⁶) ''where &alpha; is the concentration of &alpha;-phermone in nM''<br />
<br />
The initial amounts are given in the table below.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Species<br />
!Amount<br />
|-<br />
|Fus3<br />
|200<br />
|-<br />
|Fus3PP<br />
|0<br />
|-<br />
|Ste12<br />
|200<br />
|-<br />
|Fus3Ste12<br />
|0<br />
|-<br />
|Bar1<br />
|200<br />
|-<br />
|aBar1<br />
|0<br />
|}<br />
<br />
A .zip file of the model can be downloaded [[Media:NTNU_Trondheim_Yeast.zip|here]]. The original file is in .xml format and can be opened with the Cain software[http://cain.sourceforge.net].<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/ModelTeam:NTNU Trondheim/Model2012-09-26T23:26:18Z<p>Oyas: /* Model of the trigger system for mating in yeast for RHIT */</p>
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<h1>Modelling <small>Stochastic simulations of the genetic circuit</small></h1><br />
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__TOC__<br />
==Overview==<br />
<br />
To get a better understanding of the dynamics of the system, we made a model of the system using the Cain software[http://cain.sourceforge.net] for stochastic simulations. While stochastic simulations are more computationally demanding than deterministic models based on solving ODE's, they allow for random fluctuations that may have a big impact on the system in a cell[http://dx.doi.org/10.1038/nrg1615].<br />
<br />
As we want our system to react to two different signals, three promoters were required. One to respond to lactate, one to respond to low oxygen and a third to respond to signal molecules controlled by the two other promoters. The last promoter would then control cell lysis. For lactate sensing, we have adapted the lld promoter of ''E. coli'', the vgb promoter from ''Vitreoscilla'' is used for the oxygen while the lux promoter from ''Vibrio fischeri'' was used for lysis control.<br />
<br />
The model can be divided into three parts; the lld promoter, the vgb promoter and the lux promoter. Each of these systems was first modelled separately. This made it easier to observe the effect of changing individual parameters and make reasonable estimates when experimental data were not available. The final model contained 66 reactions and 46 parameters, many of which were estimates. However, we experienced that working with the model and observing how parameter changes influenced protein expressions gave valuable insight into the system.<br />
<br />
Stochastic models, as opposed to deterministic models, describe the presence of species with the number of molecules. For small molecules, in this case oxygen and lactate, this gives very high numbers. For example; a 1 mmol concentration of lactate gives about 400000 molecules in the cell assuming a cell volume of 0.7 µm³. This many molecules are computationally demanding to keep track of and the diffusion of these molecules through the cell membrane is relatively fast[http://dx.doi.org/10.1002/bit.260320308]. To simplify, lower, constant concentrations and higher propensity functions were used. A possible problem with this simplification is greater fluctuations than a more realistic model.<br />
<br />
In general, the models seems to be too sensitive with small stimulations giving very large effects. A possible reason is that the values we used for the translation rates are too high, as these are estimates. Another possibility is that the propensity for mRNA decay are more different between deterministic models, as in the references, and stochastic models than expected. This was especially the case for the lux promoter.<br />
<br />
All the equations in the model assumed mass action. For the details and parameters used in the model, see [[Team:NTNU_Trondheim/Equations|Equations]].<br />
<br />
A .zip file of the full model can be downloaded [[Media:NTNU_Trondheim_Full.zip|here]].<br />
<br />
==Lld promoter==<br />
In ''E. coli'', the Lld promoter regulates the metabolism of l-Lactate[http://dx.doi.org/10.1128/JB.02013-07]. Two proteins involved in metabolism as well as the regulatory protein LldR is induced by the presence of lactate. LldR in solution will form dimers that will bind to two sites on the promoter DNA. This will block RNA polymerase from binding to the DNA, possibly with a looping structure[http://dx.doi.org/10.1128/JB.02013-07 [4<nowiki>]</nowiki>]. If l-Lactate is present in sufficient quantities, it will bind to the LldR dimer and cause a reconformation of the complex. This activates the promoter. A similar system is found in Corynebacterium Glutamicum[http://dx.doi.org/10.1128/JB.01147-07]. <br />
<br />
We isolated the promoter part of the operon and coupled it to expression of the LuxI protein from V. Ficheri[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0061]. As LldR is already present in ''E. Coli'', it was assumed to have an initial concentration as well as a constant production and degradation. A more realistic system could have included a self regulating feedback system, but this would further complicate the model without necessarily increasing the descriptive accuracy.<br />
<br />
Binding to the promoter was modelled as LldR in solution forming dimers that could bind to the promoter. Lactate would then bind to the dimer, either in solution or bound to DNA. This gave a quite reasonable behaviour in the presence or absence of lactate, but other paths are also possible. We were not able to find any detailed description of the mechanism.<br />
<br />
The plots show the production of the LuxI protein as a response to the presence of lactate. The plot on the left shows a system with a quite low concentration of lactate, 10 in the model. Under these conditions, only a few, less than 5, promoters are activated and, as result, the LuxI concentration remains low.The plot on the right shows the response to a 100 times larger lactate concentration. Even though the number of active promoters remains relatively low, the constant production of mRNA leads to a large production of LuxI.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_LldrLow.png|thumb|center|450px|Figure 1. Response of lld promoter to a lactate level of 10]]<br />
|[[File:NTNU_Trondheim_Model_LldrHigh.png|thumb|center|450px|Figure 2. Response of lld promoter to a lactate level of 1000]]<br />
|}<br />
<br />
==Vgb promoter==<br />
''Vitreoscilla'' is a gram-negative, aerobic bacteria[http://dx.doi.org/10.1016/j.micres.2005.03.004]. To facilitate continued oxygen uptake, it produces bacterial hemoglobin in low oxygen environments. This production is regulated by the vgb promoter[http://partsregistry.org/wiki/index.php?title=Part:BBa_K561001] that has little or low expression in aerobic and anaerobic conditions, but is strongly expressed in microaerobic conditions[http://jb.asm.org/content/171/11/5995]. This behaviour is caused by two proteins; Fnr and ArcA[http://dx.doi.org/10.1016/j.micres.2005.03.004 [7<nowiki>]</nowiki>]. <br />
<br />
Both of these proteins are also found in ''E. Coli'' and are involved in the regulation of at least 20 different processes in the cell[http://dx.doi.org/10.1111/j.1574-6968.1990.tb04109.x]. Fnr contains an [2Fe-2S]²⁺ cluster that is directly oxidized in the precence of oxygen[http://dx.doi.org/10.1016/S0014-5793(97)01219-2]. This was modelled as the unoxidized protein binding to the promoter, thus activating it. In the precense of high oxygen concentrations, however, it would be converted to an oxidized form that did not bind to the promoter.<br />
<br />
For the ArcA protein, the system is more complicated. In anaerobic conditions, a membrane protein called ArcB will autophosphorylate. This will in turn phosphorylate ArcA which can then inhibit the vgb promoter[http://dx.doi.org/10.1126/science.1059361]. To simplify the model, this was simulated the same way as the Fnr protein with direct oxidation, but with a higher oxidation propensity and the binding of the reduced protein caused inhibition, not activation. This gave a qualitative description of the system with little expression with high or very low oxygen concentrations and activation at intermediate levels.<br />
<br />
In the figures below, the LuxR production and amount of active promoter is plotted for three different oxygen levels; 10, 250 and 2000. For the levels with low induction, the amount of protein remains relatively low around 100. In the high induction case, however, the concentration rapidly increases to over 900. Even for the high induction case, the amount of activated promoter remains relatively low.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_VgbLow.png|thumb|center|450px|Figure 3. Response of vgb promoter to an oxygen level of 10]]<br />
|[[File:NTNU_Trondheim_Model_VgbHigh.png|thumb|center|450px|Figure 4. Response of vgb promoter to an oxygen level of 250]]<br />
|}<br />
[[File:NTNU_Trondheim_Model_VgbVeryHigh.png|thumb|center|450px|Figure 5. Response of vgb promoter to an oxygen level of 1000]]<br />
<br />
==Lux promoter==<br />
To control the production of holin, which causes cell lysis, see full model, the lux promoter from ''Vibrio fischeri'' were used. V. fischeri lives in the light emitting organs of certain marine animals. When floating freely in the ocean, the bacteria do not produce light, however, in the light organs, the bacteria will achive high concentrations and start to luminesce[http://dx.doi.org/10.1146/annurev.micro.50.1.727]. Luminescense is controlled by the lux promoter that is activated by a complex of the protein LuxR and homoserine lactone (HSL). <br />
<br />
HSL is a relatively small signalling molecule that is formed by the reaction between S-adenosylmethionine (SAM) and 3-oxo-hexanoyl-CoA (hex). This reaction is catalyzed bu the LuxI enzyme. LuxI is constitutively expressed in the cells, but HSL will diffuse through the cell membrane and not reach high enough concentrations to activate the promoter if there is no HSL in the environment surrounding the cell. At high cell concentrations the amount of HSL, however, a buildup of HSL will activate the promoter. The lux promoter has been adapted as a biobrick and used successfully in ''E. coli''[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062].<br />
<br />
To model the production of HSL, a constant production and degradation of SAM and hex was assumed. In the presence of LuxI a complex would form and HSL would be produced, consuming the substrates. The diffusion of HSL was modelled as a first order mass action equation with HSL going to the null space.<br />
<br />
In the cell, two LuxR proteins will form a complex with two HSL molecules. This complex will then bind to a palindromic site and activate the promoter. In the model, the reaction was described as two HSL and two proteins reacting directly and forming a complex that activated the promoter. The results are shown in the figure below. In the figure to the left, a constant production of 0.001 s⁻¹ was assumed for both LuxR and LuxI. This gave an expression of Holin, the compound that cause lysis, see full model, with a maximum about 700. This was quite high, but less than the lethal amount of about a 1000[http://dx.doi.org/10.1146/annurev.micro.54.1.799]. After increasing the expression to 0.01 s⁻¹, the amount increased to about 5500. Again, the amount of activated promoter were quite low, indicating a too high transcription and translation rate.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_LuxBoxLow.png|thumb|center|450px|Figure 6. Response of lux promoter to a LuxI and LuxR production rate of 0.001 s⁻¹]]<br />
|[[File:NTNU_Trondheim_Model_LuxBoxHigh.png|thumb|center|450px|Figure 7. Response of lux promoter to a LuxI and LuxR production rate of 0.01 s⁻¹]]<br />
|}<br />
<br />
==Full model==<br />
In the full model, the previous parts were put together and made to control the lysis genes. The lysis genes[http://partsregistry.org/wiki/index.php/Part:BBa_K112808] are adapted from the T4 phage. T4 infect gram negative bacteria and cause lysis in them. This is caused by two compounds; a membrane protein called holin and an endolysin that degrades cell walls. The endolysin itself is relatively harmless for the bacteria, as it cannot pass through the inner membrane[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>]. To facilitate lysis, holin is required as it creates pores in the inner membrane. About 1000 holin molecules per cell is required for lysis[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>].<br />
<br />
For the phage, there is an optimal delay between infection and lysis, as it takes time to assemble viruses in the cell. To delay lysis, the genes for an antiholin is also present in the gene cassette. Antiholin forms a complex with holin, thus disabling the creation of pores. An infected bacteria will behave normally up until lysis begins[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>].<br />
<br />
The figures below describe the behaviour of the system under various conditions. Figure 8 shows the system with a relatively low oxygen level of 100 and a high lactate level of 1000. The antiholin will cause a delay in lysis, but holin will reach lethal levels in about an hour. Figure 9 shows the response to oxygen levels of 2000 and lactate levels of 10. In this case, there is still holin production, but most of it is complexed with antiholin and it does not reach lethal levels.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_HolinHolinHigh.png|thumb|center|450px|Figure 8. Production rate of holin at oxygen levels of 100 and lactate levels of 1000]]<br />
|[[File:NTNU_Trondheim_Model_HolinHolinLow.png|thumb|center|450px|Figure 9. Production rate of holin at oxygen levels of 2000 and lactate levels of 10]]<br />
|}<br />
<br />
Figure 10 shows the production of LuxI and LuxR with maximally inducing oxygen levels, about 250, but no lactate. There is much LuxR being produced, but no LuxI and thus no HSL. In the model, it turned out to be difficult to turn the system completely off, as long as there were lactate present, even in small amounts. This is probably not realistic, as V. fischeri has a constant production of both LuxR and LuxI without inducing luminescence. <br />
<br />
The last plot shows the response to high oxygen levels; 5000, and high lactate levels; 100. this gives a response similiar to the one in Figure 9 were holin is produced, but forms a complex with antiholin and does not reach large enough quantities to cause lysis.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_HighOxNoLact.png|thumb|center|450px|Figure 10. Production rate of LuxR and LuxI at oxygen levels of 250 and no lactate]]<br />
|[[File:NTNU_Trondheim_Model_LactVHighOx.png|thumb|center|450px|Figure 11. Production rate of holin at oxygen levels of 5000 and lactate levels of 100]]<br />
|}<br />
<br />
==Modelling for RHIT==<br />
<br />
Our part of the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaboration</a> with RHiT was helping them with stochastic modelling of the trigger system for mating in yeast. The full mechanism has been quite well studied, but it is very complicated[http://dx.doi.org/10.1002/yea.1122]. In RHiTs [[Team:RHIT/Modeling|model]], the final steps of the mechanism is activation of the Ste12 protein by the Fus3 enzyme. To simplify the model, production of Fus3 in the model was described by a sigmoid curve found in experiments[http://dx.doi.org/10.1038/nature08946] with respect to the concentration of &alpha;-pheromone. Inactive Ste12 was quickly activated by the presence of Fus3, so the outcome of active Ste12 followed a similar sigmoid curve, giving the expected switch behaviour. The resulting plot is shown in Figure 1. Each point is the average of 100 trajectories with the error bars indicating one standard deviation.<br />
<br />
[[File:NTNU_Trondheim_YeastAlpha.png|thumb|center|450px|Figure 1. Amount of activated Ste12 at steady state as a response to &alpha;-pheromone concentrations. Error bars show one standard deviation.]]<br />
<br />
The equations used in the model are given in the table below. The parameters are taken from[http://dx.doi.org/10.1002/yea.1122 [1<nowiki>]</nowiki>] and are modelled using mass action solvers, except Fus3 &rarr; Fus3PP, which use a sigmoid function. Timesteps in the model are minutes.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Reaction<br />
!Propensity<br />
!Comment<br />
|-<br />
|Fus3 &rarr; Fus3PP<br />
|*<br />
|Activation of Fus3<br />
|-<br />
|Fus3PP &rarr; Fus3<br />
|150<br />
|Deactivation of Fus3<br />
|-<br />
|Fus3PP + Ste12 &rarr; Fus3Ste12<br />
|18<br />
|Activation of Ste12 through complexation with Fus3<br />
|-<br />
|Fus3Ste12 &rarr; Fus3PP + Ste12<br />
|10<br />
|Deactivation of Ste12 by release of Fus3<br />
|-<br />
|Bar1 + Fus3Ste12 &rarr; aBar1 + Fus3Ste12<br />
|0.1<br />
|Activation of Bar1 enzyme<br />
|-<br />
|aBar1 &rarr; Bar1<br />
|0.1<br />
|Deactivation of Bar1<br />
|-<br />
|aBar1 &rarr; ø<br />
|0.01<br />
|Export of active Bar1<br />
|}<br />
''* The function for Fus3 activation is given by'' 200*&alpha;⁶/(&alpha;⁶ + 150⁶) ''where &alpha; is the concentration of &alpha;-phermone in nM''<br />
<br />
The initial amounts are given in the table below.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Species<br />
!Amount<br />
|-<br />
|Fus3<br />
|200<br />
|-<br />
|Fus3PP<br />
|0<br />
|-<br />
|Ste12<br />
|200<br />
|-<br />
|Fus3Ste12<br />
|0<br />
|-<br />
|Bar1<br />
|200<br />
|-<br />
|aBar1<br />
|0<br />
|}<br />
<br />
A .zip file of the model can be downloaded [[Media:NTNU_Trondheim_Yeast.zip|here]]. The original file is in .xml format and can be opened with the Cain software[http://cain.sourceforge.net].<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/ModelTeam:NTNU Trondheim/Model2012-09-26T23:25:59Z<p>Oyas: </p>
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<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Modelling <small>Stochastic simulations of the genetic circuit</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
==Overview==<br />
<br />
To get a better understanding of the dynamics of the system, we made a model of the system using the Cain software[http://cain.sourceforge.net] for stochastic simulations. While stochastic simulations are more computationally demanding than deterministic models based on solving ODE's, they allow for random fluctuations that may have a big impact on the system in a cell[http://dx.doi.org/10.1038/nrg1615].<br />
<br />
As we want our system to react to two different signals, three promoters were required. One to respond to lactate, one to respond to low oxygen and a third to respond to signal molecules controlled by the two other promoters. The last promoter would then control cell lysis. For lactate sensing, we have adapted the lld promoter of ''E. coli'', the vgb promoter from ''Vitreoscilla'' is used for the oxygen while the lux promoter from ''Vibrio fischeri'' was used for lysis control.<br />
<br />
The model can be divided into three parts; the lld promoter, the vgb promoter and the lux promoter. Each of these systems was first modelled separately. This made it easier to observe the effect of changing individual parameters and make reasonable estimates when experimental data were not available. The final model contained 66 reactions and 46 parameters, many of which were estimates. However, we experienced that working with the model and observing how parameter changes influenced protein expressions gave valuable insight into the system.<br />
<br />
Stochastic models, as opposed to deterministic models, describe the presence of species with the number of molecules. For small molecules, in this case oxygen and lactate, this gives very high numbers. For example; a 1 mmol concentration of lactate gives about 400000 molecules in the cell assuming a cell volume of 0.7 µm³. This many molecules are computationally demanding to keep track of and the diffusion of these molecules through the cell membrane is relatively fast[http://dx.doi.org/10.1002/bit.260320308]. To simplify, lower, constant concentrations and higher propensity functions were used. A possible problem with this simplification is greater fluctuations than a more realistic model.<br />
<br />
In general, the models seems to be too sensitive with small stimulations giving very large effects. A possible reason is that the values we used for the translation rates are too high, as these are estimates. Another possibility is that the propensity for mRNA decay are more different between deterministic models, as in the references, and stochastic models than expected. This was especially the case for the lux promoter.<br />
<br />
All the equations in the model assumed mass action. For the details and parameters used in the model, see [[Team:NTNU_Trondheim/Equations|Equations]].<br />
<br />
A .zip file of the full model can be downloaded [[Media:NTNU_Trondheim_Full.zip|here]].<br />
<br />
==Lld promoter==<br />
In ''E. coli'', the Lld promoter regulates the metabolism of l-Lactate[http://dx.doi.org/10.1128/JB.02013-07]. Two proteins involved in metabolism as well as the regulatory protein LldR is induced by the presence of lactate. LldR in solution will form dimers that will bind to two sites on the promoter DNA. This will block RNA polymerase from binding to the DNA, possibly with a looping structure[http://dx.doi.org/10.1128/JB.02013-07 [4<nowiki>]</nowiki>]. If l-Lactate is present in sufficient quantities, it will bind to the LldR dimer and cause a reconformation of the complex. This activates the promoter. A similar system is found in Corynebacterium Glutamicum[http://dx.doi.org/10.1128/JB.01147-07]. <br />
<br />
We isolated the promoter part of the operon and coupled it to expression of the LuxI protein from V. Ficheri[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0061]. As LldR is already present in ''E. Coli'', it was assumed to have an initial concentration as well as a constant production and degradation. A more realistic system could have included a self regulating feedback system, but this would further complicate the model without necessarily increasing the descriptive accuracy.<br />
<br />
Binding to the promoter was modelled as LldR in solution forming dimers that could bind to the promoter. Lactate would then bind to the dimer, either in solution or bound to DNA. This gave a quite reasonable behaviour in the presence or absence of lactate, but other paths are also possible. We were not able to find any detailed description of the mechanism.<br />
<br />
The plots show the production of the LuxI protein as a response to the presence of lactate. The plot on the left shows a system with a quite low concentration of lactate, 10 in the model. Under these conditions, only a few, less than 5, promoters are activated and, as result, the LuxI concentration remains low.The plot on the right shows the response to a 100 times larger lactate concentration. Even though the number of active promoters remains relatively low, the constant production of mRNA leads to a large production of LuxI.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_LldrLow.png|thumb|center|450px|Figure 1. Response of lld promoter to a lactate level of 10]]<br />
|[[File:NTNU_Trondheim_Model_LldrHigh.png|thumb|center|450px|Figure 2. Response of lld promoter to a lactate level of 1000]]<br />
|}<br />
<br />
==Vgb promoter==<br />
''Vitreoscilla'' is a gram-negative, aerobic bacteria[http://dx.doi.org/10.1016/j.micres.2005.03.004]. To facilitate continued oxygen uptake, it produces bacterial hemoglobin in low oxygen environments. This production is regulated by the vgb promoter[http://partsregistry.org/wiki/index.php?title=Part:BBa_K561001] that has little or low expression in aerobic and anaerobic conditions, but is strongly expressed in microaerobic conditions[http://jb.asm.org/content/171/11/5995]. This behaviour is caused by two proteins; Fnr and ArcA[http://dx.doi.org/10.1016/j.micres.2005.03.004 [7<nowiki>]</nowiki>]. <br />
<br />
Both of these proteins are also found in ''E. Coli'' and are involved in the regulation of at least 20 different processes in the cell[http://dx.doi.org/10.1111/j.1574-6968.1990.tb04109.x]. Fnr contains an [2Fe-2S]²⁺ cluster that is directly oxidized in the precence of oxygen[http://dx.doi.org/10.1016/S0014-5793(97)01219-2]. This was modelled as the unoxidized protein binding to the promoter, thus activating it. In the precense of high oxygen concentrations, however, it would be converted to an oxidized form that did not bind to the promoter.<br />
<br />
For the ArcA protein, the system is more complicated. In anaerobic conditions, a membrane protein called ArcB will autophosphorylate. This will in turn phosphorylate ArcA which can then inhibit the vgb promoter[http://dx.doi.org/10.1126/science.1059361]. To simplify the model, this was simulated the same way as the Fnr protein with direct oxidation, but with a higher oxidation propensity and the binding of the reduced protein caused inhibition, not activation. This gave a qualitative description of the system with little expression with high or very low oxygen concentrations and activation at intermediate levels.<br />
<br />
In the figures below, the LuxR production and amount of active promoter is plotted for three different oxygen levels; 10, 250 and 2000. For the levels with low induction, the amount of protein remains relatively low around 100. In the high induction case, however, the concentration rapidly increases to over 900. Even for the high induction case, the amount of activated promoter remains relatively low.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_VgbLow.png|thumb|center|450px|Figure 3. Response of vgb promoter to an oxygen level of 10]]<br />
|[[File:NTNU_Trondheim_Model_VgbHigh.png|thumb|center|450px|Figure 4. Response of vgb promoter to an oxygen level of 250]]<br />
|}<br />
[[File:NTNU_Trondheim_Model_VgbVeryHigh.png|thumb|center|450px|Figure 5. Response of vgb promoter to an oxygen level of 1000]]<br />
<br />
==Lux promoter==<br />
To control the production of holin, which causes cell lysis, see full model, the lux promoter from ''Vibrio fischeri'' were used. V. fischeri lives in the light emitting organs of certain marine animals. When floating freely in the ocean, the bacteria do not produce light, however, in the light organs, the bacteria will achive high concentrations and start to luminesce[http://dx.doi.org/10.1146/annurev.micro.50.1.727]. Luminescense is controlled by the lux promoter that is activated by a complex of the protein LuxR and homoserine lactone (HSL). <br />
<br />
HSL is a relatively small signalling molecule that is formed by the reaction between S-adenosylmethionine (SAM) and 3-oxo-hexanoyl-CoA (hex). This reaction is catalyzed bu the LuxI enzyme. LuxI is constitutively expressed in the cells, but HSL will diffuse through the cell membrane and not reach high enough concentrations to activate the promoter if there is no HSL in the environment surrounding the cell. At high cell concentrations the amount of HSL, however, a buildup of HSL will activate the promoter. The lux promoter has been adapted as a biobrick and used successfully in ''E. coli''[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0062].<br />
<br />
To model the production of HSL, a constant production and degradation of SAM and hex was assumed. In the presence of LuxI a complex would form and HSL would be produced, consuming the substrates. The diffusion of HSL was modelled as a first order mass action equation with HSL going to the null space.<br />
<br />
In the cell, two LuxR proteins will form a complex with two HSL molecules. This complex will then bind to a palindromic site and activate the promoter. In the model, the reaction was described as two HSL and two proteins reacting directly and forming a complex that activated the promoter. The results are shown in the figure below. In the figure to the left, a constant production of 0.001 s⁻¹ was assumed for both LuxR and LuxI. This gave an expression of Holin, the compound that cause lysis, see full model, with a maximum about 700. This was quite high, but less than the lethal amount of about a 1000[http://dx.doi.org/10.1146/annurev.micro.54.1.799]. After increasing the expression to 0.01 s⁻¹, the amount increased to about 5500. Again, the amount of activated promoter were quite low, indicating a too high transcription and translation rate.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_LuxBoxLow.png|thumb|center|450px|Figure 6. Response of lux promoter to a LuxI and LuxR production rate of 0.001 s⁻¹]]<br />
|[[File:NTNU_Trondheim_Model_LuxBoxHigh.png|thumb|center|450px|Figure 7. Response of lux promoter to a LuxI and LuxR production rate of 0.01 s⁻¹]]<br />
|}<br />
<br />
==Full model==<br />
In the full model, the previous parts were put together and made to control the lysis genes. The lysis genes[http://partsregistry.org/wiki/index.php/Part:BBa_K112808] are adapted from the T4 phage. T4 infect gram negative bacteria and cause lysis in them. This is caused by two compounds; a membrane protein called holin and an endolysin that degrades cell walls. The endolysin itself is relatively harmless for the bacteria, as it cannot pass through the inner membrane[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>]. To facilitate lysis, holin is required as it creates pores in the inner membrane. About 1000 holin molecules per cell is required for lysis[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>].<br />
<br />
For the phage, there is an optimal delay between infection and lysis, as it takes time to assemble viruses in the cell. To delay lysis, the genes for an antiholin is also present in the gene cassette. Antiholin forms a complex with holin, thus disabling the creation of pores. An infected bacteria will behave normally up until lysis begins[http://dx.doi.org/10.1146/annurev.micro.54.1.799 [15<nowiki>]</nowiki>].<br />
<br />
The figures below describe the behaviour of the system under various conditions. Figure 8 shows the system with a relatively low oxygen level of 100 and a high lactate level of 1000. The antiholin will cause a delay in lysis, but holin will reach lethal levels in about an hour. Figure 9 shows the response to oxygen levels of 2000 and lactate levels of 10. In this case, there is still holin production, but most of it is complexed with antiholin and it does not reach lethal levels.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_HolinHolinHigh.png|thumb|center|450px|Figure 8. Production rate of holin at oxygen levels of 100 and lactate levels of 1000]]<br />
|[[File:NTNU_Trondheim_Model_HolinHolinLow.png|thumb|center|450px|Figure 9. Production rate of holin at oxygen levels of 2000 and lactate levels of 10]]<br />
|}<br />
<br />
Figure 10 shows the production of LuxI and LuxR with maximally inducing oxygen levels, about 250, but no lactate. There is much LuxR being produced, but no LuxI and thus no HSL. In the model, it turned out to be difficult to turn the system completely off, as long as there were lactate present, even in small amounts. This is probably not realistic, as V. fischeri has a constant production of both LuxR and LuxI without inducing luminescence. <br />
<br />
The last plot shows the response to high oxygen levels; 5000, and high lactate levels; 100. this gives a response similiar to the one in Figure 9 were holin is produced, but forms a complex with antiholin and does not reach large enough quantities to cause lysis.<br />
<br />
{| style="text-align:center; margin: 1em auto 1em auto; padding: 0 10px;" cellpadding="10px"<br />
|[[File:NTNU_Trondheim_Model_HighOxNoLact.png|thumb|center|450px|Figure 10. Production rate of LuxR and LuxI at oxygen levels of 250 and no lactate]]<br />
|[[File:NTNU_Trondheim_Model_LactVHighOx.png|thumb|center|450px|Figure 11. Production rate of holin at oxygen levels of 5000 and lactate levels of 100]]<br />
|}<br />
<br />
==Model of the trigger system for mating in yeast for RHIT==<br />
<br />
Our part of the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaboration</a> with RHiT was helping them with stochastic modelling of the trigger system for mating in yeast. The full mechanism has been quite well studied, but it is very complicated[http://dx.doi.org/10.1002/yea.1122]. In RHiTs [[Team:RHIT/Modeling|model]], the final steps of the mechanism is activation of the Ste12 protein by the Fus3 enzyme. To simplify the model, production of Fus3 in the model was described by a sigmoid curve found in experiments[http://dx.doi.org/10.1038/nature08946] with respect to the concentration of &alpha;-pheromone. Inactive Ste12 was quickly activated by the presence of Fus3, so the outcome of active Ste12 followed a similar sigmoid curve, giving the expected switch behaviour. The resulting plot is shown in Figure 1. Each point is the average of 100 trajectories with the error bars indicating one standard deviation.<br />
<br />
[[File:NTNU_Trondheim_YeastAlpha.png|thumb|center|450px|Figure 1. Amount of activated Ste12 at steady state as a response to &alpha;-pheromone concentrations. Error bars show one standard deviation.]]<br />
<br />
The equations used in the model are given in the table below. The parameters are taken from[http://dx.doi.org/10.1002/yea.1122 [1<nowiki>]</nowiki>] and are modelled using mass action solvers, except Fus3 &rarr; Fus3PP, which use a sigmoid function. Timesteps in the model are minutes.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Reaction<br />
!Propensity<br />
!Comment<br />
|-<br />
|Fus3 &rarr; Fus3PP<br />
|*<br />
|Activation of Fus3<br />
|-<br />
|Fus3PP &rarr; Fus3<br />
|150<br />
|Deactivation of Fus3<br />
|-<br />
|Fus3PP + Ste12 &rarr; Fus3Ste12<br />
|18<br />
|Activation of Ste12 through complexation with Fus3<br />
|-<br />
|Fus3Ste12 &rarr; Fus3PP + Ste12<br />
|10<br />
|Deactivation of Ste12 by release of Fus3<br />
|-<br />
|Bar1 + Fus3Ste12 &rarr; aBar1 + Fus3Ste12<br />
|0.1<br />
|Activation of Bar1 enzyme<br />
|-<br />
|aBar1 &rarr; Bar1<br />
|0.1<br />
|Deactivation of Bar1<br />
|-<br />
|aBar1 &rarr; ø<br />
|0.01<br />
|Export of active Bar1<br />
|}<br />
''* The function for Fus3 activation is given by'' 200*&alpha;⁶/(&alpha;⁶ + 150⁶) ''where &alpha; is the concentration of &alpha;-phermone in nM''<br />
<br />
The initial amounts are given in the table below.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Species<br />
!Amount<br />
|-<br />
|Fus3<br />
|200<br />
|-<br />
|Fus3PP<br />
|0<br />
|-<br />
|Ste12<br />
|200<br />
|-<br />
|Fus3Ste12<br />
|0<br />
|-<br />
|Bar1<br />
|200<br />
|-<br />
|aBar1<br />
|0<br />
|}<br />
<br />
A .zip file of the model can be downloaded [[Media:NTNU_Trondheim_Yeast.zip|here]]. The original file is in .xml format and can be opened with the Cain software[http://cain.sourceforge.net].<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/YeastTeam:NTNU Trondheim/Yeast2012-09-26T23:23:43Z<p>Oyas: </p>
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<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
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<div class="container"><br />
<div class="page-header-top"><br />
<h1>Collaboration with RHiT; Yeast modelling <small>Stochastic simulations of the genetic circuit</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
Our part of the collaboration with RHiT was helping them with stochastic modelling of the trigger system for mating in yeast. The full mechanism has been quite well studied, but it is very complicated[http://dx.doi.org/10.1002/yea.1122]. In RHiTs [[Team:RHIT/Modeling|model]], the final steps of the mechanism is activation of the Ste12 protein by the Fus3 enzyme. To simplify the model, production of Fus3 in the model was described by a sigmoid curve found in experiments[http://dx.doi.org/10.1038/nature08946] with respect to the concentration of &alpha;-pheromone. Inactive Ste12 was quickly activated by the presence of Fus3, so the outcome of active Ste12 followed a similar sigmoid curve, giving the expected switch behaviour. The resulting plot is shown in Figure 1. Each point is the average of 100 trajectories with the error bars indicating one standard deviation.<br />
<br />
[[File:NTNU_Trondheim_YeastAlpha.png|thumb|center|450px|Figure 1. Amount of activated Ste12 at steady state as a response to &alpha;-pheromone concentrations. Error bars show one standard deviation.]]<br />
<br />
The equations used in the model are given in the table below. The parameters are taken from[http://dx.doi.org/10.1002/yea.1122 [1<nowiki>]</nowiki>] and are modelled using mass action solvers, except Fus3 &rarr; Fus3PP, which use a sigmoid function. Timesteps in the model are minutes.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Reaction<br />
!Propensity<br />
!Comment<br />
|-<br />
|Fus3 &rarr; Fus3PP<br />
|*<br />
|Activation of Fus3<br />
|-<br />
|Fus3PP &rarr; Fus3<br />
|150<br />
|Deactivation of Fus3<br />
|-<br />
|Fus3PP + Ste12 &rarr; Fus3Ste12<br />
|18<br />
|Activation of Ste12 through complexation with Fus3<br />
|-<br />
|Fus3Ste12 &rarr; Fus3PP + Ste12<br />
|10<br />
|Deactivation of Ste12 by release of Fus3<br />
|-<br />
|Bar1 + Fus3Ste12 &rarr; aBar1 + Fus3Ste12<br />
|0.1<br />
|Activation of Bar1 enzyme<br />
|-<br />
|aBar1 &rarr; Bar1<br />
|0.1<br />
|Deactivation of Bar1<br />
|-<br />
|aBar1 &rarr; ø<br />
|0.01<br />
|Export of active Bar1<br />
|}<br />
''* The function for Fus3 activation is given by'' 200*&alpha;⁶/(&alpha;⁶ + 150⁶) ''where &alpha; is the concentration of &alpha;-phermone in nM''<br />
<br />
The initial amounts are given in the table below.<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|-<br />
!Species<br />
!Amount<br />
|-<br />
|Fus3<br />
|200<br />
|-<br />
|Fus3PP<br />
|0<br />
|-<br />
|Ste12<br />
|200<br />
|-<br />
|Fus3Ste12<br />
|0<br />
|-<br />
|Bar1<br />
|200<br />
|-<br />
|aBar1<br />
|0<br />
|}<br />
<br />
A .zip file of the model can be downloaded [[Media:NTNU_Trondheim_Yeast.zip|here]]. The original file is in .xml format and can be opened with the Cain software[http://cain.sourceforge.net].<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/YeastTeam:NTNU Trondheim/Yeast2012-09-26T23:21:18Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Collaboration with RHiT; Yeast modelling <small>Stochastic simulations of the genetic circuit</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
[[File:NTNU_Trondheim_YeastAlpha.png|thumb|450px|Figure 1. Amount of activated Ste12 at steady state as a response to &alpha;-pheromone concentrations. Error bars show one standard deviation.]]<br />
<br />
Our part of the collaboration with RHiT was helping them with stochastic modelling of the trigger system for mating in yeast. The full mechanism has been quite well studied, but it is very complicated[http://dx.doi.org/10.1002/yea.1122]. In RHiTs [[Team:RHIT/Modeling|model]], the final steps of the mechanism is activation of the Ste12 protein by the Fus3 enzyme. To simplify the model, production of Fus3 in the model was described by a sigmoid curve found in experiments[http://dx.doi.org/10.1038/nature08946] with respect to the concentration of &alpha;-pheromone. Inactive Ste12 was quickly activated by the presence of Fus3, so the outcome of active Ste12 followed a similar sigmoid curve, giving the expected switch behaviour. The resulting plot is shown in Figure 1. Each point is the average of 100 trajectories with the error bars indicating one standard deviation.<br />
<br />
The equations used in the model are given in the table below. The parameters are taken from[http://dx.doi.org/10.1002/yea.1122 [1<nowiki>]</nowiki>] and are modelled using mass action solvers, except Fus3 &rarr; Fus3PP, which use a sigmoid function. Timesteps in the model are minutes.<br />
<br />
<br />
{|border="1"<br />
|-<br />
!Reaction<br />
!Propensity<br />
!Comment<br />
|-<br />
|Fus3 &rarr; Fus3PP<br />
|*<br />
|Activation of Fus3<br />
|-<br />
|Fus3PP &rarr; Fus3<br />
|150<br />
|Deactivation of Fus3<br />
|-<br />
|Fus3PP + Ste12 &rarr; Fus3Ste12<br />
|18<br />
|Activation of Ste12 through complexation with Fus3<br />
|-<br />
|Fus3Ste12 &rarr; Fus3PP + Ste12<br />
|10<br />
|Deactivation of Ste12 by release of Fus3<br />
|-<br />
|Bar1 + Fus3Ste12 &rarr; aBar1 + Fus3Ste12<br />
|0.1<br />
|Activation of Bar1 enzyme<br />
|-<br />
|aBar1 &rarr; Bar1<br />
|0.1<br />
|Deactivation of Bar1<br />
|-<br />
|aBar1 &rarr; ø<br />
|0.01<br />
|Export of active Bar1<br />
|}<br />
''* The function for Fus3 activation is given by'' 200*&alpha;⁶/(&alpha;⁶ + 150⁶) ''where &alpha; is the concentration of &alpha;-phermone in nM''<br />
<br />
<br />
The initial amounts are given in the table below.<br />
<br />
{|border="1"<br />
|-<br />
!Species<br />
!Amount<br />
|-<br />
|Fus3<br />
|200<br />
|-<br />
|Fus3PP<br />
|0<br />
|-<br />
|Ste12<br />
|200<br />
|-<br />
|Fus3Ste12<br />
|0<br />
|-<br />
|Bar1<br />
|200<br />
|-<br />
|aBar1<br />
|0<br />
|}<br />
<br />
A .zip file of the model can be downloaded [[Media:NTNU_Trondheim_Yeast.zip|here]]. The original file is in .xml format and can be opened with the Cain software[http://cain.sourceforge.net].<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T23:13:05Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
==Introduction==<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
==Colicin (<partinfo>BBa_K822002</partinfo>)==<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{|<br />
|[[File:Colicin2.png|500px]]<br />
|-<br />
|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)<br />
|}<br />
<br />
{|<br />
|[[File:Colicin1.png|500px]]<br />
|-<br />
|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added. <br />
<br />
==YFP generator (<partinfo>BBa_K822003</partinfo>)==<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
==Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)==<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
{|border="0" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|thumb|center|300px|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.]]<br />
|style="vertical-align: top;"|[[File:RBS+LacI+term-gel.PNG|thumb|center|450px|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. ]]<br />
|}<br />
<br />
==lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)==<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
==ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)==<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T23:11:28Z<p>Oyas: /* BioBrick parts */</p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
==Introduction==<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
==Colicin (<partinfo>BBa_K822002</partinfo>)==<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{|<br />
|[[File:Colicin2.png|500px]]<br />
|-<br />
|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)<br />
|}<br />
<br />
{|<br />
|[[File:Colicin1.png|500px]]<br />
|-<br />
|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added. <br />
<br />
<br />
==YFP generator (<partinfo>BBa_K822003</partinfo>)==<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
==Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)==<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
{|border="0" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|thumb|center|300px|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.]]<br />
|style="vertical-align: top;"|[[File:RBS+LacI+term-gel.PNG|thumb|center|450px|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. ]]<br />
|}<br />
<br />
==lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)==<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
<br />
==ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)==<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T23:10:59Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
==BioBrick parts==<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
==Colicin (<partinfo>BBa_K822002</partinfo>)==<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{|<br />
|[[File:Colicin2.png|500px]]<br />
|-<br />
|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)<br />
|}<br />
<br />
{|<br />
|[[File:Colicin1.png|500px]]<br />
|-<br />
|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added. <br />
<br />
<br />
==YFP generator (<partinfo>BBa_K822003</partinfo>)==<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
==Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)==<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
{|border="0" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|thumb|center|300px|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.]]<br />
|style="vertical-align: top;"|[[File:RBS+LacI+term-gel.PNG|thumb|center|450px|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. ]]<br />
|}<br />
<br />
==lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)==<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
<br />
==ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)==<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T23:07:50Z<p>Oyas: /* Regulative LacI generator (BBa_K822004) */</p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
<br />
==Experiments and results==<br />
<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
<br />
===Colicin (<partinfo>BBa_K822002</partinfo>)===<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{|<br />
|[[File:Colicin2.png|500px]]<br />
|-<br />
|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)<br />
|}<br />
<br />
{|<br />
|[[File:Colicin1.png|500px]]<br />
|-<br />
|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added. <br />
<br />
<br />
===YFP generator (<partinfo>BBa_K822003</partinfo>)===<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
===Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)===<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
{|border="0" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|thumb|center|300px|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.]]<br />
|style="vertical-align: top;"|[[File:RBS+LacI+term-gel.PNG|thumb|center|450px|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. ]]<br />
|}<br />
<br />
===lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)===<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
<br />
===ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)===<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T23:01:15Z<p>Oyas: /* Regulative LacI generator (BBa_K822004) */</p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
<br />
==Experiments and results==<br />
<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
<br />
===Colicin (<partinfo>BBa_K822002</partinfo>)===<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{|<br />
|[[File:Colicin2.png|500px]]<br />
|-<br />
|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)<br />
|}<br />
<br />
{|<br />
|[[File:Colicin1.png|500px]]<br />
|-<br />
|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added. <br />
<br />
<br />
===YFP generator (<partinfo>BBa_K822003</partinfo>)===<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
===Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)===<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
{|border="0"<br />
|[[File:Testkutt_BBa_K292006.png|thumb|center|x300px|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.]]<br />
|[[File:RBS+LacI+term-gel.PNG|thumb|center|x300px|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. ]]<br />
|}<br />
<br />
===lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)===<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
<br />
===ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)===<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T22:59:21Z<p>Oyas: /* YFP generator (BBa_K822003) */</p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
<br />
==Experiments and results==<br />
<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
<br />
===Colicin (<partinfo>BBa_K822002</partinfo>)===<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{|<br />
|[[File:Colicin2.png|500px]]<br />
|-<br />
|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)<br />
|}<br />
<br />
{|<br />
|[[File:Colicin1.png|500px]]<br />
|-<br />
|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added. <br />
<br />
<br />
===YFP generator (<partinfo>BBa_K822003</partinfo>)===<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
[[File:YFP_fluorescence.png|thumb|center|500px||Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP]]<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
===Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)===<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|x300px]]<br />
|[[File:RBS+LacI+term-gel.PNG|x300px]]<br />
|-<br />
|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.<br />
|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. <br />
|}<br />
<br />
===lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)===<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
<br />
===ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)===<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_ResultsTeam:NTNU Trondheim/Experiments and Results2012-09-26T22:58:28Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Experiments and results <small>How we tested our components and what we found out</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
</html><br />
__TOC__<br />
<br />
==Experiments and results==<br />
<br />
To make a genetic circuit releasing colicin as a response to a low oxygen level and a high lactate level, we needed several biobricks. For a detailed list of all biobricks present in our construct, see the [https://2012.igem.org/Team:NTNU_Trondheim/Parts parts page].<br />
<br />
Most of the biobricks we decided to use were already present in the registry, but we also needed biobricks with certain properties that were not present in the registry. These we had to make ourselves. The new bricks we made, and which we also characterized, are the following; <br />
<br />
* a protein coding brick for colicin E1, <br />
<br />
* a YFP-generator, a regulative LacI-generator, which is also an improvement of an already existing biobrick, <br />
<br />
* the lld promotor + RBS from ''E.coli'', <br />
<br />
* the lld promotor + RBS from ''C.glutamicum''.<br />
<br />
This page will focus on the biobricks we have made, how we made them, and how we have characterized them to show that they work.<br />
<br />
<br />
===Colicin (<partinfo>BBa_K822002</partinfo>)===<br />
<br />
Colicin is the protein we have chosen as toxin in our bacterial anti-cancer-kamikaze device. We amplified the brick using <partinfo>BBa_K150009</partinfo> as template. The brick contains protein coding sequences both for Colicin E1 and for colicin immunity protein. The following primers were used:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Colicin fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGatggaaaccgcggtagcgta<br />
|-<br />
|Colicin rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcgatggtccctccctgaa<br />
|}<br />
<br />
<br />
To test if the our colicin brick worked, we cloned it together with a constitutive promoter + RBS <partinfo>BBa_K081005</partinfo>. Then, we grew overnight cultures with the Promoter+RBS+Colicin construct, and also with a negative control. The negative control were different in different experiments, but were always cells containing a non-expressing plasmid with ampicillin resistance, since the plasmid colicin was tested in, also had ampicillin resistance. <br />
After about 24 hours, LB was replaced with breaking buffer and the cells were sonicated. The lysed cells were centrifuged at 16000 g for 15 minutes to remove cell fragments, and the 3 ml lysate was added to a new 10 ml culture of newly inoculated ''E.coli'' cells with ampicillin resistance, but without the colicin immunity protein. Samples were taken regularly, and OD was measured. <br />
We performed two experiments; one experiment with two parallel cell cultures, where one was containing colicin and the other buffer (1), and one experiment with two parallels of cells with colicin lysate added, and one with lysate of non-colicin-producing cells added (2). The latter experiment was performed to prove that no other proteins expressed by the cells inhibited growth in other cells. The results of the experiments are given below:<br />
<br />
{|<br />
|[[File:Colicin2.png|500px]]<br />
|-<br />
|OD measured over time in cell cultures with added colicin lysate (red dots) and buffer (blue dots)<br />
|}<br />
<br />
{|<br />
|[[File:Colicin1.png|500px]]<br />
|-<br />
|OD measured over time in two parallel cell cultures with added colicin lysate (red and purple dots) and lysate of non-colicin-producing cells (blue dots)<br />
|}<br />
<br />
Both experiments show that the cells without added colicin lysate has a significantly higher growth rate than the cells where colicin lysate was added. <br />
<br />
<br />
===YFP generator (<partinfo>BBa_K822003</partinfo>)===<br />
<br />
We made the YFP generator to verify that the YFP coding sequence worked, in order to use it in experiments verifying that the Vgb promoter worked. The YFP generator was cloned together from a constitutive promoter + RBS (<partinfo>BBa_K081005</partinfo>), YFP (<partinfo>BBa_E0030</partinfo>) and a double terminator (<partinfo>BBa_B0015</partinfo>).<br />
<br />
To verify that the YFP generator worked, cells containing the YFP generator plasmid and cells containing plasmids with YFP without promoter, RBS or terminator (only BBa_E0030), and plasmids containing Vgb+RBS+YFP+terminator (<partinfo>BBa_K561001</partinfo>+<partinfo>BBa_E0030</partinfo>) was grown overnight, and fluorescence measured. The emission wavelength was 544 nm, and 514 nm was used for exitation. Four parallel measurements were carried out. The result of the measurement can be seen below:<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:YFP_fluorescence.png|500px]]<br />
|-<br />
|Fluorescence measured in Constitutive promoter+RBS+YFP+terminator, YFP, and Vgb+RBS+YFP<br />
|}<br />
<br />
Even though we were not able to prove that the Vgb promoter works, we proved that the YFP generator works, as the fluorescence divided by OD is much higher for cells containing this biobrick, than for example for cells containing plasmids with only YFP.<br />
<br />
===Regulative LacI generator (<partinfo>BBa_K822004</partinfo>)===<br />
<br />
We made this brick in an effort to improve an already existing biobrick. The brick we wanted to improve was <partinfo>BBa_K292006</partinfo>. The NTNU iGEM team 2011 tried to use this brick in their stress sensor, but did not get it to work. They also tried to test-cut it and investigate the fragments using gel electrophoresis, however the resulting fragments were not as expected. This is why we thought of this biobrick as a suitable candidate for improvement. <br />
Since it is a composite part, we cloned it together again from scratch, using RBS (<partinfo>BBa_B0030</partinfo>), LacI (<partinfo>BBa_C0012</partinfo>) and a double terminator (<partinfo>BBa_B0014</partinfo>).<br />
<br />
When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [https://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.<br />
<br />
Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
|[[File:Testkutt_BBa_K292006.png|x300px]]<br />
|[[File:RBS+LacI+term-gel.PNG|x300px]]<br />
|-<br />
|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.<br />
|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense. <br />
|}<br />
<br />
===lld promoter + RBS from ''E.coli'' (<partinfo>BBa_K822000</partinfo>)===<br />
<br />
The two criteria we wanted fulfilled to initiate lysis and subsequent release of colicin were a low oxygen level and a high lactate level. A promoter activated by low oxygen level was already present in the registry (microaerobic Vgb promoter, <partinfo>BBa_K561001</partinfo>), but we found no suitable lactate-induced promoter. Therefore, we decided to convert the promotor regulating the lldPRD operon in ''E.coli'' into a biobrick, and to use this biobrick in our project [[http://www.ncbi.nlm.nih.gov/pubmed/18263722 1]].<br />
<br />
The primers used to amplify the sequence are given below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld EcR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGcacattcctataggccgagtaaggt<br />
|-<br />
|Plld EcR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAtgcaggtctcctggagtccacgc<br />
|}<br />
<br />
The capitalized letters of the primer sequences corresponds to the biobrick prefix and suffix. As template, we used the ''E.coli K12'' genome sequence provided by the NCBI nucleotide database [[http://www.ncbi.nlm.nih.gov/nuccore/U00096.2 2]]. These primers in combination with the genome from ''E.coli'' K12 MG1655, yielded a biobrick consisting of the lld promoter including RBS (We called this brick Plld EcR, Ec because it is amplified from ''E.coli'', R because it contains RBS).<br />
<br />
We did not have sufficient time to test the Plld EcR biobrick, but it was sent to sequencing in the official shipping plasmid, pSB1C3, and the sequencing result had a 100 % match with the theoretical sequence of the amplified Plld + RBS sequence in pSB1C3.<br />
<br />
<br />
===ldhA promoter + RBS from ''C.glutamicum'' (<partinfo>BBa_K822001</partinfo>)===<br />
<br />
We also amplified the ldhA promoter from ''Corynebacterium glutamicum''. This has similar properties as the lld promoter from ''E.coli'', so this promoter was also a candidate to being used as the lactate inducable promoter in our project.<br />
The ldhA promoter was amplified using the genome of ''C.glutamicum'' ATC 13032 as template, and the primers below:<br />
<br />
<br />
{|class="table table-bordered table-hover" style="margin: 1em auto 1em auto; width: auto;"<br />
!Primer<br />
!Sequence<br />
|-<br />
|Plld CgR fwd<br />
|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGctctgttgcttaaat<br />
|-<br />
|Plld CgR rev<br />
|GTTTCTTCCTGCAGCGGCCGCTACTAGTAggtgacctcttctctgaaacgg<br />
|}<br />
<br />
The promoter has not been properly characterized, but sequencing indicated a 100 % match with the theoretical sequence.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
</div></div></html><br />
{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/AchievementsTeam:NTNU Trondheim/Achievements2012-09-26T22:56:06Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
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<h1>Achievements <small>How we have fulfilled the judging criteria</small></h1><br />
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<div class="container main-container"><br />
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<table class="table table-bordered table-left"> <br />
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<th> Medal<br />
</th><th> Criteria<br />
</th><th> How we have fulfilled the criteria<br />
</th><br />
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</thead><br />
<tbody><br />
<tr><br />
<th rowspan="5" style="vertical-align: middle;" ><i class="icon-certificate" style="color: #A67D3D;font-size:24pt;"></i><br/>Bronze<br />
</th><td>Team registration<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> The team has been registered. See our <a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">official team profile</a>.<br />
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<tr><br />
<td>Complete Judging form<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have completed the <a href="https://igem.org/2012_Judging_Form?id=822" target="_blank">judging form</a>.<br />
</td></tr><br />
<tr><br />
<td>Team Wiki<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have set up a <a href="https://2012.igem.org/Team:NTNU_Trondheim">team wiki</a>.<br />
</td></tr><br />
<tr><br />
<td>Present a poster and a talk at the iGEM Jamboree<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have prepared a poster and a presentation for the European jamboree.<br />
</td></tr><br />
<tr><br />
<td>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> device.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a <a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822000">lldPRD operon promoter + RBS from <i>Escherichia coli</i>.</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822001">ldhA promoter + RBS from <i>Corynebacterium glutamicum</i> </a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted a <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>.<br/><br />
</td></tr><br />
<tr><br />
<th rowspan="2" style="vertical-align: middle;"><i class="icon-certificate" style="color: #E6E8FA;font-size:24pt;"></i><br/>Silver<br />
</td><td>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected<br />
</td><td rowspan="2"><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that two of our BioBrick parts work as expected, the <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> part and the <a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a>. We have characterized them and entered the information in the Parts Registry. Please take a look at our <a href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Experiments and results</a> section. <br />
</td></tr><br />
<tr><br />
<td>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.<br />
</td></tr><br />
<tr> <br />
<th rowspan="3" style="vertical-align: middle;"><i class="icon-certificate" style="color: #D9D919;font-size:24pt;"></i><br/> Gold<br />
</th><td>Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have improved the <a href="http://partsregistry.org/Part:BBa_K292006">Strong RBS + LacI repressor + double terminator</a> part and entered the information of the corrected part, the <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>, into a new registry page. We sequenced the original BioBrick part, showing that it was not correct, reassembled it and confirmed that the sequence of the improved part was correct.<br />
</td></tr><br />
<tr><br />
<td> Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaborated with the Rose-Hulman Institute of Technology iGEM team</a>. We constructed a stochastic model of their system and they helped us with the characterization of one of our lactate activated promoters, the pLLD promoter from <i>Escherichia coli</i>.<br />
</td></tr><br />
<tr><br />
<td> Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have created an online tool for facilitating collaboration between iGEM teams. We have called it the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker" target="_blank">iGEM Matchmaker</a><br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/AchievementsTeam:NTNU Trondheim/Achievements2012-09-26T22:46:20Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Achievements <small>How we have fulfilled the judging criteria</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
<br />
<div class="row "><br />
<div class="span12"><br />
<br />
<table class="table table-bordered table-left"> <br />
<thead><br />
<tr><br />
<th> Medal<br />
</th><th> Criteria<br />
</th><th> How we have fulfilled the criteria<br />
</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<th rowspan="5" style="vertical-align: middle;" ><i class="icon-certificate" style="color: #A67D3D;font-size:24pt;"></i><br/>Bronze<br />
</th><td>Team registration<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> The team has been registered. See our <a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">official team profile</a>.<br />
</td></tr><br />
<tr><br />
<td>Complete Judging form<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have completed the <a href="https://igem.org/2012_Judging_Form?id=822" target="_blank">judging form</a>.<br />
</td></tr><br />
<tr><br />
<td>Team Wiki<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have set up a <a href="https://2012.igem.org/Team:NTNU_Trondheim">team wiki</a>.<br />
</td></tr><br />
<tr><br />
<td>Present a poster and a talk at the iGEM Jamboree<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have prepared a poster and a presentation for the European jamboree.<br />
</td></tr><br />
<tr><br />
<td>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> device.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a<a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822000">lldPRD operon promoter + RBS from <i>Escherichia coli</i>.</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822001">ldhA promoter + RBS from <i>Corynebacterium glutamicum</i> </a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted a <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>.<br/><br />
</td></tr><br />
<tr><br />
<th rowspan="2" style="vertical-align: middle;"><i class="icon-certificate" style="color: #E6E8FA;font-size:24pt;"></i><br/>Silver<br />
</td><td>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected<br />
</td><td rowspan="2"><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br />
</td></tr><br />
<tr><br />
<td>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.<br />
</td></tr><br />
<tr> <br />
<th rowspan="3" style="vertical-align: middle;"><i class="icon-certificate" style="color: #D9D919;font-size:24pt;"></i><br/> Gold<br />
</th><td>Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have improved the <a href="http://partsregistry.org/Part:BBa_K292006">Strong RBS + LacI repressor + double terminator</a> part and entered the information of the corrected part, the <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>, into a new registry page. We sequenced the original BioBrick part, showing that it was not correct, reassembled it and confirmed that the sequence of the improved part was correct.<br />
</td></tr><br />
<tr><br />
<td> Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaborated with the Rose-Hulman Institute of Technology iGEM team</a>. We constructed a stochastic model of their system and they helped us with the characterization of one of our lactate activated promoters, the pLLD promoter from <i>Escherichia coli</i>.<br />
</td></tr><br />
<tr><br />
<td> Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have created an online tool for facilitating collaboration between iGEM teams. We have called it the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker" target="_blank">iGEM Matchmaker</a><br />
</td></tr><br />
</tbody><br />
</table><br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/AchievementsTeam:NTNU Trondheim/Achievements2012-09-26T22:39:15Z<p>Oyas: </p>
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<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Achievements <small>How we have fulfilled the judging criteria</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
<br />
<div class="row "><br />
<div class="span12"><br />
<br />
<table class="table table-bordered table-left"> <br />
<thead><br />
<tr><br />
<th> Medal<br />
</th><th> Criteria<br />
</th><th> How we have fulfilled the criteria<br />
</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<th rowspan="5" style="vertical-align: middle;" ><i class="icon-certificate" style="color: #A67D3D;font-size:24pt;"></i><br/>Bronze<br />
</th><td>Team registration<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> The team has been registered. See our <a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">official team profile</a>.<br />
</td></tr><br />
<tr><br />
<td>Complete Judging form<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have completed the <a href="https://igem.org/2012_Judging_Form?id=822" target="_blank">judging form</a>.<br />
</td></tr><br />
<tr><br />
<td>Team Wiki<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have set up a <a href="https://2012.igem.org/Team:NTNU_Trondheim">team wiki</a>.<br />
</td></tr><br />
<tr><br />
<td>Present a poster and a talk at the iGEM Jamboree<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have prepared a poster and a presentation for the European jamboree.<br />
</td></tr><br />
<tr><br />
<td>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> device.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a<a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822000">lldPRD operon promoter + RBS from <i>Escherichia coli</i>.</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822001">ldhA promoter + RBS from <i>Corynebacterium glutamicum</i> </a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted a <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>.<br/><br />
</td></tr><br />
<tr><br />
<th rowspan="2" style="vertical-align: middle;"><i class="icon-certificate" style="color: #E6E8FA;font-size:24pt;"></i><br/>Silver<br />
</td><td>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected<br />
</td><td rowspan="2"><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br />
</td></tr><br />
<tr><br />
<td>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.<br />
</td></tr><br />
<tr> <br />
<th rowspan="3" style="vertical-align: middle;"><i class="icon-certificate" style="color: #D9D919;font-size:24pt;"></i><br/> Gold<br />
</th><td>Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have improved the <a href="http://partsregistry.org/Part:BBa_K292006">Strong RBS + LacI repressor + double terminator</a> part and entered the information of the corrected part, the <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>, into a new registry page. We sequenced the original BioBrick part, showing that it was not correct, reassembled it and confirmed that the sequence of the improved part was correct.<br />
</td></tr><br />
<tr><br />
</td></tr><br />
<tr><br />
<td> Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaborated with the Rose-Hulman Institute of Technology iGEM team</a>. We constructed a stochastic model of their system and they helped us with the characterization of one of our lactate activated promoters, the pLLD promoter from <i>Escherichia coli</i>.<br />
</td></tr><br />
</tbody><br />
</table><br />
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</div><br />
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</div><br />
</html><br />
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{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
<html><br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/AchievementsTeam:NTNU Trondheim/Achievements2012-09-26T22:34:20Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
<html><br />
<div class="container"><br />
<div class="page-header-top"><br />
<h1>Achievements <small>How we have fulfilled the judging criteria</small></h1><br />
</div><br />
</div><br />
<br />
<br />
<div class="container main-container"><br />
<br />
<div class="row "><br />
<div class="span12"><br />
<br />
<table class="table table-bordered table-left"> <br />
<thead><br />
<tr><br />
<th> Medal<br />
</th><th> Criteria<br />
</th><th> How we have fulfilled the criteria<br />
</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<th rowspan="5" style="vertical-align: middle;" ><i class="icon-certificate" style="color: #A67D3D;font-size:24pt;"></i><br/>Bronze<br />
</th><td>Team registration<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> The team has been registered. See our <a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">official team profile</a>.<br />
</td></tr><br />
<tr><br />
<td>Complete Judging form<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have completed the <a href="https://igem.org/2012_Judging_Form?id=822" target="_blank">judging form</a>.<br />
</td></tr><br />
<tr><br />
<td>Team Wiki<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have set up a <a href="https://2012.igem.org/Team:NTNU_Trondheim">team wiki</a>.<br />
</td></tr><br />
<tr><br />
<td>Present a poster and a talk at the iGEM Jamboree<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have prepared a poster and a presentation for the European jamboree.<br />
</td></tr><br />
<tr><br />
<td>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> device.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a<a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822000">lldPRD operon promoter + RBS from <i>Escherichia coli</i>.</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822001">ldhA promoter + RBS from <i>Corynebacterium glutamicum</i> </a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted a <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>.<br/><br />
</td></tr><br />
<tr><br />
<th rowspan="2" style="vertical-align: middle;"><i class="icon-certificate" style="color: #E6E8FA;font-size:24pt;"></i><br/>Silver<br />
</td><td>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected<br />
</td><td rowspan="2"><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br />
</td></tr><br />
<tr><br />
<td>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.<br />
</td></tr><br />
<tr> <br />
<th rowspan="3" style="vertical-align: middle;"><i class="icon-certificate" style="color: #D9D919;font-size:24pt;"></i><br/> Gold<br />
</th><td>Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have improved the <a href="http://partsregistry.org/Part:BBa_K292006">Strong RBS + LacI repressor + double terminator</a> part and entered the information of the corrected part, the <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>, into a new registry page. We sequenced the original BioBrick part, showing that it was not correct, reassembled it and confirmed that the sequence of the improved part was correct.<br />
</td></tr><br />
<tr><br />
</td></tr><br />
<tr><br />
<td> Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaborated with the Rose-Hulman Institute of Technology iGEM team</a>. We constructed a stochastic model of their system and they helped us with the characterization of one of our lactate activated promoters, the pLLD promoter from <i>Escherichia coli</i>.<br />
</td></tr><br />
<tr><br />
</td></tr><br />
<tr><br />
<td> Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have created an online tool for facilitating collaboration between iGEM teams. We have called it the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker" target="_blank">iGEM Matchmaker</a><br />
</td></tr><br />
</tbody><br />
</table><br />
<br />
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{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/AchievementsTeam:NTNU Trondheim/Achievements2012-09-26T22:33:18Z<p>Oyas: </p>
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<h1>Achievements <small>How we have fulfilled the judging criteria</small></h1><br />
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</div><br />
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<br />
<div class="container main-container"><br />
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<tr><br />
<th> Medal<br />
</th><th> Criteria<br />
</th><th> How we have fulfilled the criteria<br />
</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<th rowspan="5" style="vertical-align: middle;" ><i class="icon-certificate" style="color: #A67D3D;font-size:24pt;"></i><br/>Bronze<br />
</th><td>Team registration<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> The team has been registered. See our <a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">official team profile</a>.<br />
</td></tr><br />
<tr><br />
<td>Complete Judging form<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have completed the <a href="https://igem.org/2012_Judging_Form?id=822" target="_blank">judging form</a>.<br />
</td></tr><br />
<tr><br />
<td>Team Wiki<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have set up a <a href="https://2012.igem.org/Team:NTNU_Trondheim">team wiki</a>.<br />
</td></tr><br />
<tr><br />
<td>Present a poster and a talk at the iGEM Jamboree<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have prepared a poster and a presentation for the European jamboree.<br />
</td></tr><br />
<tr><br />
<td>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> device.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a<a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822000">lldPRD operon promoter + RBS from <i>Escherichia coli</i>.</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822001">ldhA promoter + RBS from <i>Corynebacterium glutamicum</i> </a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted a <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>.<br/><br />
</td></tr><br />
<tr><br />
<th rowspan="2" style="vertical-align: middle;"><i class="icon-certificate" style="color: #E6E8FA;font-size:24pt;"></i><br/>Silver<br />
</td><td>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected<br />
</td><td rowspan="2"><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br />
</td></tr><br />
<tr><br />
<td>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.<br />
</td></tr><br />
<tr> <br />
<th rowspan="3" style="vertical-align: middle;"><i class="icon-certificate" style="color: #D9D919;font-size:24pt;"></i><br/> Gold<br />
</th><td>Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have improved the <a href="http://partsregistry.org/Part:BBa_K292006">Strong RBS + LacI repressor + double terminator</a> part and entered the information of the corrected part, the <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>, into a new registry page. We sequenced the original BioBrick part, showing that it was not correct, reassembled it and confirmed that the sequence of the improved part was correct.<br />
</td></tr><br />
<tr><br />
</td></tr><br />
<tr><br />
<td> Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaborated with the Rose-Hulman Institute of Technology iGEM team</a>. We constructed a stochastic model of their system and they helped us with the characterization of one of our lactate activated promoters, the pLLD promoter from <i>Escherichia coli</i>.<br />
</td></tr><br />
<tr><br />
<td> Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have created an online tool for facilitating collaboration between iGEM teams. We have called it the <a href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker" target="_blank">iGEM Matchmaker</a><br />
</td></tr><br />
</tbody><br />
</table><br />
<br />
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{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/AchievementsTeam:NTNU Trondheim/Achievements2012-09-26T22:26:47Z<p>Oyas: </p>
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<h1>Achievements <small>How we have fulfilled the judging criteria</small></h1><br />
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</div><br />
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<br />
<div class="container main-container"><br />
<br />
<div class="row "><br />
<div class="span12"><br />
<br />
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<th> Medal<br />
</th><th> Criteria<br />
</th><th> How we have fulfilled the criteria<br />
</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<th rowspan="5" style="vertical-align: middle;" ><i class="icon-certificate" style="color: #A67D3D;font-size:24pt;"></i><br/>Bronze<br />
</th><td>Team registration<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> The team has been registered. See our <a href="https://igem.org/Team.cgi?year=2012&team_name=NTNU_Trondheim" target="_blank">official team profile</a>.<br />
</td></tr><br />
<tr><br />
<td>Complete Judging form<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have completed the <a href="https://igem.org/2012_Judging_Form?id=822" target="_blank">judging form</a>.<br />
</td></tr><br />
<tr><br />
<td>Team Wiki<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have set up a <a href="https://2012.igem.org/Team:NTNU_Trondheim">team wiki</a>.<br />
</td></tr><br />
<tr><br />
<td>Present a poster and a talk at the iGEM Jamboree<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have prepared a poster and a presentation for the European jamboree.<br />
</td></tr><br />
<tr><br />
<td>At least one new submitted and well-characterized standard BioBrick Part or Device. A new application of and outstanding documentation (quantitative data showing the Part’s/ Device’s function) of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry also counts.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> device.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted and characterized a<a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822000">lldPRD operon promoter + RBS from <i>Escherichia coli</i>.</a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted the <a href="http://partsregistry.org/Part:BBa_K822001">ldhA promoter + RBS from <i>Corynebacterium glutamicum</i> </a>.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have submitted a <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>.<br/><br />
</td></tr><br />
<tr><br />
<th rowspan="2" style="vertical-align: middle;"><i class="icon-certificate" style="color: #E6E8FA;font-size:24pt;"></i><br/>Silver<br />
</td><td>Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected<br />
</td><td rowspan="2"><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822002">colicin E1 + immunoprotein</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br/><br />
<i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have demonstrated that our <a href="http://partsregistry.org/Part:BBa_K822003">constitutive YFP generator</a> part works as expected, characterized it and entered the information in the Parts Registry.<br/><br />
</td></tr><br />
<tr><br />
<td>Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.<br />
</td></tr><br />
<tr> <br />
<th rowspan="3" style="vertical-align: middle;"><i class="icon-certificate" style="color: #D9D919;font-size:24pt;"></i><br/> Gold<br />
</th><td>Improve the function of an existing BioBrick Part or Device (created by another team or your own institution in a previous year) and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry), and don't forget to create a new registry page for the improved part.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have improved the <a href="http://partsregistry.org/Part:BBa_K292006">Strong RBS + LacI repressor + double terminator</a> part and entered the information of the corrected part, the <a href="http://partsregistry.org/Part:BBa_K822004">LacI generator</a>, into a new registry page. We sequenced the original BioBrick part, showing that it was not correct, reassembled it and confirmed that the sequence of the improved part was correct.<br />
</td></tr><br />
<tr><br />
</td></tr><br />
<tr><br />
<td> Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system.<br />
</td><td><i class="icon-check" style="color:#46a546; font-size: 14pt;"></i> We have <a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">collaborated with the Rose-Hulman Institute of Technology iGEM team</a>. We constructed a stochastic model of their system and they helped us with the characterization of one of our lactate activated promoters, the pLLD promoter from <i>Escherichia coli</i>.<br />
</td></tr><br />
</tbody><br />
</table><br />
<br />
</div><br />
<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/FooterTeam:NTNU Trondheim/Templates/Footer2012-09-26T22:25:35Z<p>Oyas: </p>
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</html></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/FooterTeam:NTNU Trondheim/Templates/Footer2012-09-26T22:23:25Z<p>Oyas: </p>
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// The display texts for the counters if only one<br />
labels1: ['Year', 'Month', 'Week', 'Day', 'Hour', 'Minute', 'Second'],<br />
compactLabels: ['y', 'm', 'w', 'd'], // The compact texts for the counters<br />
whichLabels: null, // Function to determine which labels to use<br />
timeSeparator: ':', // Separator for time periods<br />
isRTL: false // True for right-to-left languages, false for left-to-right<br />
};<br />
this._defaults = {<br />
until: null, // new Date(year, mth - 1, day, hr, min, sec) - date/time to count down to<br />
// or numeric for seconds offset, or string for unit offset(s):<br />
// 'Y' years, 'O' months, 'W' weeks, 'D' days, 'H' hours, 'M' minutes, 'S' seconds<br />
since: null, // new Date(year, mth - 1, day, hr, min, sec) - date/time to count up from<br />
// or numeric for seconds offset, or string for unit offset(s):<br />
// 'Y' years, 'O' months, 'W' weeks, 'D' days, 'H' hours, 'M' minutes, 'S' seconds<br />
timezone: null, // The timezone (hours or minutes from GMT) for the target times,<br />
// or null for client local<br />
serverSync: null, // A function to retrieve the current server time for synchronisation<br />
format: 'dHMS', // Format for display - upper case for always, lower case only if non-zero,<br />
// 'Y' years, 'O' months, 'W' weeks, 'D' days, 'H' hours, 'M' minutes, 'S' seconds<br />
layout: '', // Build your own layout for the countdown<br />
compact: false, // True to display in a compact format, false for an expanded one<br />
significant: 0, // The number of periods with values to show, zero for all<br />
description: '', // The description displayed for the countdown<br />
expiryUrl: '', // A URL to load upon expiry, replacing the current page<br />
expiryText: '', // Text to display upon expiry, replacing the countdown<br />
alwaysExpire: false, // True to trigger onExpiry even if never counted down<br />
onExpiry: null, // Callback when the countdown expires -<br />
// receives no parameters and 'this' is the containing division<br />
onTick: null, // Callback when the countdown is updated -<br />
// receives int[7] being the breakdown by period (based on format)<br />
// and 'this' is the containing division<br />
tickInterval: 1 // Interval (seconds) between onTick callbacks<br />
};<br />
$.extend(this._defaults, this.regional['']);<br />
this._serverSyncs = [];<br />
// Shared timer for all countdowns<br />
function timerCallBack(timestamp) {<br />
var drawStart = (timestamp || new Date().getTime());<br />
if (drawStart - animationStartTime >= 1000) {<br />
$.countdown._updateTargets();<br />
animationStartTime = drawStart;<br />
}<br />
requestAnimationFrame(timerCallBack);<br />
}<br />
var requestAnimationFrame = window.requestAnimationFrame || window.webkitRequestAnimationFrame ||<br />
window.mozRequestAnimationFrame || window.oRequestAnimationFrame ||<br />
window.msRequestAnimationFrame || null; // this is when we expect a fall-back to setInterval as it's much more fluid<br />
var animationStartTime = 0;<br />
if (!requestAnimationFrame) {<br />
setInterval(function() { $.countdown._updateTargets(); }, 980); // Fall back to good old setInterval<br />
}<br />
else {<br />
animationStartTime = window.mozAnimationStartTime || new Date().getTime();<br />
requestAnimationFrame(timerCallBack);<br />
}<br />
}<br />
<br />
var PROP_NAME = 'countdown';<br />
<br />
var Y = 0; // Years<br />
var O = 1; // Months<br />
var W = 2; // Weeks<br />
var D = 3; // Days<br />
var H = 4; // Hours<br />
var M = 5; // Minutes<br />
var S = 6; // Seconds<br />
<br />
$.extend(Countdown.prototype, {<br />
/* Class name added to elements to indicate already configured with countdown. */<br />
markerClassName: 'hasCountdown',<br />
<br />
/* List of currently active countdown targets. */<br />
_timerTargets: [],<br />
<br />
/* Override the default settings for all instances of the countdown widget.<br />
@param options (object) the new settings to use as defaults */<br />
setDefaults: function(options) {<br />
this._resetExtraLabels(this._defaults, options);<br />
extendRemove(this._defaults, options || {});<br />
},<br />
<br />
/* Convert a date/time to UTC.<br />
@param tz (number) the hour or minute offset from GMT, e.g. +9, -360<br />
@param year (Date) the date/time in that timezone or<br />
(number) the year in that timezone<br />
@param month (number, optional) the month (0 - 11) (omit if year is a Date)<br />
@param day (number, optional) the day (omit if year is a Date)<br />
@param hours (number, optional) the hour (omit if year is a Date)<br />
@param mins (number, optional) the minute (omit if year is a Date)<br />
@param secs (number, optional) the second (omit if year is a Date)<br />
@param ms (number, optional) the millisecond (omit if year is a Date)<br />
@return (Date) the equivalent UTC date/time */<br />
UTCDate: function(tz, year, month, day, hours, mins, secs, ms) {<br />
if (typeof year == 'object' && year.constructor == Date) {<br />
ms = year.getMilliseconds();<br />
secs = year.getSeconds();<br />
mins = year.getMinutes();<br />
hours = year.getHours();<br />
day = year.getDate();<br />
month = year.getMonth();<br />
year = year.getFullYear();<br />
}<br />
var d = new Date();<br />
d.setUTCFullYear(year);<br />
d.setUTCDate(1);<br />
d.setUTCMonth(month || 0);<br />
d.setUTCDate(day || 1);<br />
d.setUTCHours(hours || 0);<br />
d.setUTCMinutes((mins || 0) - (Math.abs(tz) < 30 ? tz * 60 : tz));<br />
d.setUTCSeconds(secs || 0);<br />
d.setUTCMilliseconds(ms || 0);<br />
return d;<br />
},<br />
<br />
/* Convert a set of periods into seconds.<br />
Averaged for months and years.<br />
@param periods (number[7]) the periods per year/month/week/day/hour/minute/second<br />
@return (number) the corresponding number of seconds */<br />
periodsToSeconds: function(periods) {<br />
return periods[0] * 31557600 + periods[1] * 2629800 + periods[2] * 604800 +<br />
periods[3] * 86400 + periods[4] * 3600 + periods[5] * 60 + periods[6];<br />
},<br />
<br />
/* Retrieve one or more settings values.<br />
@param name (string, optional) the name of the setting to retrieve<br />
or 'all' for all instance settings or omit for all default settings<br />
@return (any) the requested setting(s) */<br />
_settingsCountdown: function(target, name) {<br />
if (!name) {<br />
return $.countdown._defaults;<br />
}<br />
var inst = $.data(target, PROP_NAME);<br />
return (name == 'all' ? inst.options : inst.options[name]);<br />
},<br />
<br />
/* Attach the countdown widget to a div.<br />
@param target (element) the containing division<br />
@param options (object) the initial settings for the countdown */<br />
_attachCountdown: function(target, options) {<br />
var $target = $(target);<br />
if ($target.hasClass(this.markerClassName)) {<br />
return;<br />
}<br />
$target.addClass(this.markerClassName);<br />
var inst = {options: $.extend({}, options),<br />
_periods: [0, 0, 0, 0, 0, 0, 0]};<br />
$.data(target, PROP_NAME, inst);<br />
this._changeCountdown(target);<br />
},<br />
<br />
/* Add a target to the list of active ones.<br />
@param target (element) the countdown target */<br />
_addTarget: function(target) {<br />
if (!this._hasTarget(target)) {<br />
this._timerTargets.push(target);<br />
}<br />
},<br />
<br />
/* See if a target is in the list of active ones.<br />
@param target (element) the countdown target<br />
@return (boolean) true if present, false if not */<br />
_hasTarget: function(target) {<br />
return ($.inArray(target, this._timerTargets) > -1);<br />
},<br />
<br />
/* Remove a target from the list of active ones.<br />
@param target (element) the countdown target */<br />
_removeTarget: function(target) {<br />
this._timerTargets = $.map(this._timerTargets,<br />
function(value) { return (value == target ? null : value); }); // delete entry<br />
},<br />
<br />
/* Update each active timer target. */<br />
_updateTargets: function() {<br />
for (var i = this._timerTargets.length - 1; i >= 0; i--) {<br />
this._updateCountdown(this._timerTargets[i]);<br />
}<br />
},<br />
<br />
/* Redisplay the countdown with an updated display.<br />
@param target (jQuery) the containing division<br />
@param inst (object) the current settings for this instance */<br />
_updateCountdown: function(target, inst) {<br />
var $target = $(target);<br />
inst = inst || $.data(target, PROP_NAME);<br />
if (!inst) {<br />
return;<br />
}<br />
$target.html(this._generateHTML(inst));<br />
$target[(this._get(inst, 'isRTL') ? 'add' : 'remove') + 'Class']('countdown_rtl');<br />
var onTick = this._get(inst, 'onTick');<br />
if (onTick) {<br />
var periods = inst._hold != 'lap' ? inst._periods :<br />
this._calculatePeriods(inst, inst._show, this._get(inst, 'significant'), new Date());<br />
var tickInterval = this._get(inst, 'tickInterval');<br />
if (tickInterval == 1 || this.periodsToSeconds(periods) % tickInterval == 0) {<br />
onTick.apply(target, [periods]);<br />
}<br />
}<br />
var expired = inst._hold != 'pause' &&<br />
(inst._since ? inst._now.getTime() < inst._since.getTime() :<br />
inst._now.getTime() >= inst._until.getTime());<br />
if (expired && !inst._expiring) {<br />
inst._expiring = true;<br />
if (this._hasTarget(target) || this._get(inst, 'alwaysExpire')) {<br />
this._removeTarget(target);<br />
var onExpiry = this._get(inst, 'onExpiry');<br />
if (onExpiry) {<br />
onExpiry.apply(target, []);<br />
}<br />
var expiryText = this._get(inst, 'expiryText');<br />
if (expiryText) {<br />
var layout = this._get(inst, 'layout');<br />
inst.options.layout = expiryText;<br />
this._updateCountdown(target, inst);<br />
inst.options.layout = layout;<br />
}<br />
var expiryUrl = this._get(inst, 'expiryUrl');<br />
if (expiryUrl) {<br />
window.location = expiryUrl;<br />
}<br />
}<br />
inst._expiring = false;<br />
}<br />
else if (inst._hold == 'pause') {<br />
this._removeTarget(target);<br />
}<br />
$.data(target, PROP_NAME, inst);<br />
},<br />
<br />
/* Reconfigure the settings for a countdown div.<br />
@param target (element) the containing division<br />
@param options (object) the new settings for the countdown or<br />
(string) an individual property name<br />
@param value (any) the individual property value<br />
(omit if options is an object) */<br />
_changeCountdown: function(target, options, value) {<br />
options = options || {};<br />
if (typeof options == 'string') {<br />
var name = options;<br />
options = {};<br />
options[name] = value;<br />
}<br />
var inst = $.data(target, PROP_NAME);<br />
if (inst) {<br />
this._resetExtraLabels(inst.options, options);<br />
extendRemove(inst.options, options);<br />
this._adjustSettings(target, inst);<br />
$.data(target, PROP_NAME, inst);<br />
var now = new Date();<br />
if ((inst._since && inst._since < now) ||<br />
(inst._until && inst._until > now)) {<br />
this._addTarget(target);<br />
}<br />
this._updateCountdown(target, inst);<br />
}<br />
},<br />
<br />
/* Reset any extra labelsn and compactLabelsn entries if changing labels.<br />
@param base (object) the options to be updated<br />
@param options (object) the new option values */<br />
_resetExtraLabels: function(base, options) {<br />
var changingLabels = false;<br />
for (var n in options) {<br />
if (n != 'whichLabels' && n.match(/[Ll]abels/)) {<br />
changingLabels = true;<br />
break;<br />
}<br />
}<br />
if (changingLabels) {<br />
for (var n in base) { // Remove custom numbered labels<br />
if (n.match(/[Ll]abels[0-9]/)) {<br />
base[n] = null;<br />
}<br />
}<br />
}<br />
},<br />
<br />
/* Calculate interal settings for an instance.<br />
@param target (element) the containing division<br />
@param inst (object) the current settings for this instance */<br />
_adjustSettings: function(target, inst) {<br />
var now;<br />
var serverSync = this._get(inst, 'serverSync');<br />
var serverOffset = 0;<br />
var serverEntry = null;<br />
for (var i = 0; i < this._serverSyncs.length; i++) {<br />
if (this._serverSyncs[i][0] == serverSync) {<br />
serverEntry = this._serverSyncs[i][1];<br />
break;<br />
}<br />
}<br />
if (serverEntry != null) {<br />
serverOffset = (serverSync ? serverEntry : 0);<br />
now = new Date();<br />
}<br />
else {<br />
var serverResult = (serverSync ? serverSync.apply(target, []) : null);<br />
now = new Date();<br />
serverOffset = (serverResult ? now.getTime() - serverResult.getTime() : 0);<br />
this._serverSyncs.push([serverSync, serverOffset]);<br />
}<br />
var timezone = this._get(inst, 'timezone');<br />
timezone = (timezone == null ? -now.getTimezoneOffset() : timezone);<br />
inst._since = this._get(inst, 'since');<br />
if (inst._since != null) {<br />
inst._since = this.UTCDate(timezone, this._determineTime(inst._since, null));<br />
if (inst._since && serverOffset) {<br />
inst._since.setMilliseconds(inst._since.getMilliseconds() + serverOffset);<br />
}<br />
}<br />
inst._until = this.UTCDate(timezone, this._determineTime(this._get(inst, 'until'), now));<br />
if (serverOffset) {<br />
inst._until.setMilliseconds(inst._until.getMilliseconds() + serverOffset);<br />
}<br />
inst._show = this._determineShow(inst);<br />
},<br />
<br />
/* Remove the countdown widget from a div.<br />
@param target (element) the containing division */<br />
_destroyCountdown: function(target) {<br />
var $target = $(target);<br />
if (!$target.hasClass(this.markerClassName)) {<br />
return;<br />
}<br />
this._removeTarget(target);<br />
$target.removeClass(this.markerClassName).empty();<br />
$.removeData(target, PROP_NAME);<br />
},<br />
<br />
/* Pause a countdown widget at the current time.<br />
Stop it running but remember and display the current time.<br />
@param target (element) the containing division */<br />
_pauseCountdown: function(target) {<br />
this._hold(target, 'pause');<br />
},<br />
<br />
/* Pause a countdown widget at the current time.<br />
Stop the display but keep the countdown running.<br />
@param target (element) the containing division */<br />
_lapCountdown: function(target) {<br />
this._hold(target, 'lap');<br />
},<br />
<br />
/* Resume a paused countdown widget.<br />
@param target (element) the containing division */<br />
_resumeCountdown: function(target) {<br />
this._hold(target, null);<br />
},<br />
<br />
/* Pause or resume a countdown widget.<br />
@param target (element) the containing division<br />
@param hold (string) the new hold setting */<br />
_hold: function(target, hold) {<br />
var inst = $.data(target, PROP_NAME);<br />
if (inst) {<br />
if (inst._hold == 'pause' && !hold) {<br />
inst._periods = inst._savePeriods;<br />
var sign = (inst._since ? '-' : '+');<br />
inst[inst._since ? '_since' : '_until'] =<br />
this._determineTime(sign + inst._periods[0] + 'y' +<br />
sign + inst._periods[1] + 'o' + sign + inst._periods[2] + 'w' +<br />
sign + inst._periods[3] + 'd' + sign + inst._periods[4] + 'h' + <br />
sign + inst._periods[5] + 'm' + sign + inst._periods[6] + 's');<br />
this._addTarget(target);<br />
}<br />
inst._hold = hold;<br />
inst._savePeriods = (hold == 'pause' ? inst._periods : null);<br />
$.data(target, PROP_NAME, inst);<br />
this._updateCountdown(target, inst);<br />
}<br />
},<br />
<br />
/* Return the current time periods.<br />
@param target (element) the containing division<br />
@return (number[7]) the current periods for the countdown */<br />
_getTimesCountdown: function(target) {<br />
var inst = $.data(target, PROP_NAME);<br />
return (!inst ? null : (!inst._hold ? inst._periods :<br />
this._calculatePeriods(inst, inst._show, this._get(inst, 'significant'), new Date())));<br />
},<br />
<br />
/* Get a setting value, defaulting if necessary.<br />
@param inst (object) the current settings for this instance<br />
@param name (string) the name of the required setting<br />
@return (any) the setting's value or a default if not overridden */<br />
_get: function(inst, name) {<br />
return (inst.options[name] != null ?<br />
inst.options[name] : $.countdown._defaults[name]);<br />
},<br />
<br />
/* A time may be specified as an exact value or a relative one.<br />
@param setting (string or number or Date) - the date/time value<br />
as a relative or absolute value<br />
@param defaultTime (Date) the date/time to use if no other is supplied<br />
@return (Date) the corresponding date/time */<br />
_determineTime: function(setting, defaultTime) {<br />
var offsetNumeric = function(offset) { // e.g. +300, -2<br />
var time = new Date();<br />
time.setTime(time.getTime() + offset * 1000);<br />
return time;<br />
};<br />
var offsetString = function(offset) { // e.g. '+2d', '-4w', '+3h +30m'<br />
offset = offset.toLowerCase();<br />
var time = new Date();<br />
var year = time.getFullYear();<br />
var month = time.getMonth();<br />
var day = time.getDate();<br />
var hour = time.getHours();<br />
var minute = time.getMinutes();<br />
var second = time.getSeconds();<br />
var pattern = /([+-]?[0-9]+)\s*(s|m|h|d|w|o|y)?/g;<br />
var matches = pattern.exec(offset);<br />
while (matches) {<br />
switch (matches[2] || 's') {<br />
case 's': second += parseInt(matches[1], 10); break;<br />
case 'm': minute += parseInt(matches[1], 10); break;<br />
case 'h': hour += parseInt(matches[1], 10); break;<br />
case 'd': day += parseInt(matches[1], 10); break;<br />
case 'w': day += parseInt(matches[1], 10) * 7; break;<br />
case 'o':<br />
month += parseInt(matches[1], 10); <br />
day = Math.min(day, $.countdown._getDaysInMonth(year, month));<br />
break;<br />
case 'y':<br />
year += parseInt(matches[1], 10);<br />
day = Math.min(day, $.countdown._getDaysInMonth(year, month));<br />
break;<br />
}<br />
matches = pattern.exec(offset);<br />
}<br />
return new Date(year, month, day, hour, minute, second, 0);<br />
};<br />
var time = (setting == null ? defaultTime :<br />
(typeof setting == 'string' ? offsetString(setting) :<br />
(typeof setting == 'number' ? offsetNumeric(setting) : setting)));<br />
if (time) time.setMilliseconds(0);<br />
return time;<br />
},<br />
<br />
/* Determine the number of days in a month.<br />
@param year (number) the year<br />
@param month (number) the month<br />
@return (number) the days in that month */<br />
_getDaysInMonth: function(year, month) {<br />
return 32 - new Date(year, month, 32).getDate();<br />
},<br />
<br />
/* Determine which set of labels should be used for an amount.<br />
@param num (number) the amount to be displayed<br />
@return (number) the set of labels to be used for this amount */<br />
_normalLabels: function(num) {<br />
return num;<br />
},<br />
<br />
/* Generate the HTML to display the countdown widget.<br />
@param inst (object) the current settings for this instance<br />
@return (string) the new HTML for the countdown display */<br />
_generateHTML: function(inst) {<br />
// Determine what to show<br />
var significant = this._get(inst, 'significant');<br />
inst._periods = (inst._hold ? inst._periods :<br />
this._calculatePeriods(inst, inst._show, significant, new Date()));<br />
// Show all 'asNeeded' after first non-zero value<br />
var shownNonZero = false;<br />
var showCount = 0;<br />
var sigCount = significant;<br />
var show = $.extend({}, inst._show);<br />
for (var period = Y; period <= S; period++) {<br />
shownNonZero |= (inst._show[period] == '?' && inst._periods[period] > 0);<br />
show[period] = (inst._show[period] == '?' && !shownNonZero ? null : inst._show[period]);<br />
showCount += (show[period] ? 1 : 0);<br />
sigCount -= (inst._periods[period] > 0 ? 1 : 0);<br />
}<br />
var showSignificant = [false, false, false, false, false, false, false];<br />
for (var period = S; period >= Y; period--) { // Determine significant periods<br />
if (inst._show[period]) {<br />
if (inst._periods[period]) {<br />
showSignificant[period] = true;<br />
}<br />
else {<br />
showSignificant[period] = sigCount > 0;<br />
sigCount--;<br />
}<br />
}<br />
}<br />
var compact = this._get(inst, 'compact');<br />
var layout = this._get(inst, 'layout');<br />
var labels = (compact ? this._get(inst, 'compactLabels') : this._get(inst, 'labels'));<br />
var whichLabels = this._get(inst, 'whichLabels') || this._normalLabels;<br />
var timeSeparator = this._get(inst, 'timeSeparator');<br />
var description = this._get(inst, 'description') || '';<br />
var showCompact = function(period) {<br />
var labelsNum = $.countdown._get(inst,<br />
'compactLabels' + whichLabels(inst._periods[period]));<br />
return (show[period] ? inst._periods[period] +<br />
(labelsNum ? labelsNum[period] : labels[period]) + ' ' : '');<br />
};<br />
var showFull = function(period) {<br />
var labelsNum = $.countdown._get(inst, 'labels' + whichLabels(inst._periods[period]));<br />
return ((!significant && show[period]) || (significant && showSignificant[period]) ?<br />
'<span class="countdown_section"><span class="countdown_amount">' +<br />
inst._periods[period] + '</span><br/>' +<br />
(labelsNum ? labelsNum[period] : labels[period]) + '</span>' : '');<br />
};<br />
return (layout ? this._buildLayout(inst, show, layout, compact, significant, showSignificant) :<br />
((compact ? // Compact version<br />
'<span class="countdown_row countdown_amount' +<br />
(inst._hold ? ' countdown_holding' : '') + '">' + <br />
showCompact(Y) + showCompact(O) + showCompact(W) + showCompact(D) + <br />
(show[H] ? this._minDigits(inst._periods[H], 2) : '') +<br />
(show[M] ? (show[H] ? timeSeparator : '') +<br />
this._minDigits(inst._periods[M], 2) : '') +<br />
(show[S] ? (show[H] || show[M] ? timeSeparator : '') +<br />
this._minDigits(inst._periods[S], 2) : '') :<br />
// Full version<br />
'<span class="countdown_row countdown_show' + (significant || showCount) +<br />
(inst._hold ? ' countdown_holding' : '') + '">' +<br />
showFull(Y) + showFull(O) + showFull(W) + showFull(D) +<br />
showFull(H) + showFull(M) + showFull(S)) + '</span>' +<br />
(description ? '<span class="countdown_row countdown_descr">' + description + '</span>' : '')));<br />
},<br />
<br />
/* Construct a custom layout.<br />
@param inst (object) the current settings for this instance<br />
@param show (string[7]) flags indicating which periods are requested<br />
@param layout (string) the customised layout<br />
@param compact (boolean) true if using compact labels<br />
@param significant (number) the number of periods with values to show, zero for all<br />
@param showSignificant (boolean[7]) other periods to show for significance<br />
@return (string) the custom HTML */<br />
_buildLayout: function(inst, show, layout, compact, significant, showSignificant) {<br />
var labels = this._get(inst, (compact ? 'compactLabels' : 'labels'));<br />
var whichLabels = this._get(inst, 'whichLabels') || this._normalLabels;<br />
var labelFor = function(index) {<br />
return ($.countdown._get(inst,<br />
(compact ? 'compactLabels' : 'labels') + whichLabels(inst._periods[index])) ||<br />
labels)[index];<br />
};<br />
var digit = function(value, position) {<br />
return Math.floor(value / position) % 10;<br />
};<br />
var subs = {desc: this._get(inst, 'description'), sep: this._get(inst, 'timeSeparator'),<br />
yl: labelFor(Y), yn: inst._periods[Y], ynn: this._minDigits(inst._periods[Y], 2),<br />
ynnn: this._minDigits(inst._periods[Y], 3), y1: digit(inst._periods[Y], 1),<br />
y10: digit(inst._periods[Y], 10), y100: digit(inst._periods[Y], 100),<br />
y1000: digit(inst._periods[Y], 1000),<br />
ol: labelFor(O), on: inst._periods[O], onn: this._minDigits(inst._periods[O], 2),<br />
onnn: this._minDigits(inst._periods[O], 3), o1: digit(inst._periods[O], 1),<br />
o10: digit(inst._periods[O], 10), o100: digit(inst._periods[O], 100),<br />
o1000: digit(inst._periods[O], 1000),<br />
wl: labelFor(W), wn: inst._periods[W], wnn: this._minDigits(inst._periods[W], 2),<br />
wnnn: this._minDigits(inst._periods[W], 3), w1: digit(inst._periods[W], 1),<br />
w10: digit(inst._periods[W], 10), w100: digit(inst._periods[W], 100),<br />
w1000: digit(inst._periods[W], 1000),<br />
dl: labelFor(D), dn: inst._periods[D], dnn: this._minDigits(inst._periods[D], 2),<br />
dnnn: this._minDigits(inst._periods[D], 3), d1: digit(inst._periods[D], 1),<br />
d10: digit(inst._periods[D], 10), d100: digit(inst._periods[D], 100),<br />
d1000: digit(inst._periods[D], 1000),<br />
hl: labelFor(H), hn: inst._periods[H], hnn: this._minDigits(inst._periods[H], 2),<br />
hnnn: this._minDigits(inst._periods[H], 3), h1: digit(inst._periods[H], 1),<br />
h10: digit(inst._periods[H], 10), h100: digit(inst._periods[H], 100),<br />
h1000: digit(inst._periods[H], 1000),<br />
ml: labelFor(M), mn: inst._periods[M], mnn: this._minDigits(inst._periods[M], 2),<br />
mnnn: this._minDigits(inst._periods[M], 3), m1: digit(inst._periods[M], 1),<br />
m10: digit(inst._periods[M], 10), m100: digit(inst._periods[M], 100),<br />
m1000: digit(inst._periods[M], 1000),<br />
sl: labelFor(S), sn: inst._periods[S], snn: this._minDigits(inst._periods[S], 2),<br />
snnn: this._minDigits(inst._periods[S], 3), s1: digit(inst._periods[S], 1),<br />
s10: digit(inst._periods[S], 10), s100: digit(inst._periods[S], 100),<br />
s1000: digit(inst._periods[S], 1000)};<br />
var html = layout;<br />
// Replace period containers: {p<}...{p>}<br />
for (var i = Y; i <= S; i++) {<br />
var period = 'yowdhms'.charAt(i);<br />
var re = new RegExp('\\{' + period + '<\\}(.*)\\{' + period + '>\\}', 'g');<br />
html = html.replace(re, ((!significant && show[i]) ||<br />
(significant && showSignificant[i]) ? '$1' : ''));<br />
}<br />
// Replace period values: {pn}<br />
$.each(subs, function(n, v) {<br />
var re = new RegExp('\\{' + n + '\\}', 'g');<br />
html = html.replace(re, v);<br />
});<br />
return html;<br />
},<br />
<br />
/* Ensure a numeric value has at least n digits for display.<br />
@param value (number) the value to display<br />
@param len (number) the minimum length<br />
@return (string) the display text */<br />
_minDigits: function(value, len) {<br />
value = '' + value;<br />
if (value.length >= len) {<br />
return value;<br />
}<br />
value = '0000000000' + value;<br />
return value.substr(value.length - len);<br />
},<br />
<br />
/* Translate the format into flags for each period.<br />
@param inst (object) the current settings for this instance<br />
@return (string[7]) flags indicating which periods are requested (?) or<br />
required (!) by year, month, week, day, hour, minute, second */<br />
_determineShow: function(inst) {<br />
var format = this._get(inst, 'format');<br />
var show = [];<br />
show[Y] = (format.match('y') ? '?' : (format.match('Y') ? '!' : null));<br />
show[O] = (format.match('o') ? '?' : (format.match('O') ? '!' : null));<br />
show[W] = (format.match('w') ? '?' : (format.match('W') ? '!' : null));<br />
show[D] = (format.match('d') ? '?' : (format.match('D') ? '!' : null));<br />
show[H] = (format.match('h') ? '?' : (format.match('H') ? '!' : null));<br />
show[M] = (format.match('m') ? '?' : (format.match('M') ? '!' : null));<br />
show[S] = (format.match('s') ? '?' : (format.match('S') ? '!' : null));<br />
return show;<br />
},<br />
<br />
/* Calculate the requested periods between now and the target time.<br />
@param inst (object) the current settings for this instance<br />
@param show (string[7]) flags indicating which periods are requested/required<br />
@param significant (number) the number of periods with values to show, zero for all<br />
@param now (Date) the current date and time<br />
@return (number[7]) the current time periods (always positive)<br />
by year, month, week, day, hour, minute, second */<br />
_calculatePeriods: function(inst, show, significant, now) {<br />
// Find endpoints<br />
inst._now = now;<br />
inst._now.setMilliseconds(0);<br />
var until = new Date(inst._now.getTime());<br />
if (inst._since) {<br />
if (now.getTime() < inst._since.getTime()) {<br />
inst._now = now = until;<br />
}<br />
else {<br />
now = inst._since;<br />
}<br />
}<br />
else {<br />
until.setTime(inst._until.getTime());<br />
if (now.getTime() > inst._until.getTime()) {<br />
inst._now = now = until;<br />
}<br />
}<br />
// Calculate differences by period<br />
var periods = [0, 0, 0, 0, 0, 0, 0];<br />
if (show[Y] || show[O]) {<br />
// Treat end of months as the same<br />
var lastNow = $.countdown._getDaysInMonth(now.getFullYear(), now.getMonth());<br />
var lastUntil = $.countdown._getDaysInMonth(until.getFullYear(), until.getMonth());<br />
var sameDay = (until.getDate() == now.getDate() ||<br />
(until.getDate() >= Math.min(lastNow, lastUntil) &&<br />
now.getDate() >= Math.min(lastNow, lastUntil)));<br />
var getSecs = function(date) {<br />
return (date.getHours() * 60 + date.getMinutes()) * 60 + date.getSeconds();<br />
};<br />
var months = Math.max(0,<br />
(until.getFullYear() - now.getFullYear()) * 12 + until.getMonth() - now.getMonth() +<br />
((until.getDate() < now.getDate() && !sameDay) ||<br />
(sameDay && getSecs(until) < getSecs(now)) ? -1 : 0));<br />
periods[Y] = (show[Y] ? Math.floor(months / 12) : 0);<br />
periods[O] = (show[O] ? months - periods[Y] * 12 : 0);<br />
// Adjust for months difference and end of month if necessary<br />
now = new Date(now.getTime());<br />
var wasLastDay = (now.getDate() == lastNow);<br />
var lastDay = $.countdown._getDaysInMonth(now.getFullYear() + periods[Y],<br />
now.getMonth() + periods[O]);<br />
if (now.getDate() > lastDay) {<br />
now.setDate(lastDay);<br />
}<br />
now.setFullYear(now.getFullYear() + periods[Y]);<br />
now.setMonth(now.getMonth() + periods[O]);<br />
if (wasLastDay) {<br />
now.setDate(lastDay);<br />
}<br />
}<br />
var diff = Math.floor((until.getTime() - now.getTime()) / 1000);<br />
var extractPeriod = function(period, numSecs) {<br />
periods[period] = (show[period] ? Math.floor(diff / numSecs) : 0);<br />
diff -= periods[period] * numSecs;<br />
};<br />
extractPeriod(W, 604800);<br />
extractPeriod(D, 86400);<br />
extractPeriod(H, 3600);<br />
extractPeriod(M, 60);<br />
extractPeriod(S, 1);<br />
if (diff > 0 && !inst._since) { // Round up if left overs<br />
var multiplier = [1, 12, 4.3482, 7, 24, 60, 60];<br />
var lastShown = S;<br />
var max = 1;<br />
for (var period = S; period >= Y; period--) {<br />
if (show[period]) {<br />
if (periods[lastShown] >= max) {<br />
periods[lastShown] = 0;<br />
diff = 1;<br />
}<br />
if (diff > 0) {<br />
periods[period]++;<br />
diff = 0;<br />
lastShown = period;<br />
max = 1;<br />
}<br />
}<br />
max *= multiplier[period];<br />
}<br />
}<br />
if (significant) { // Zero out insignificant periods<br />
for (var period = Y; period <= S; period++) {<br />
if (significant && periods[period]) {<br />
significant--;<br />
}<br />
else if (!significant) {<br />
periods[period] = 0;<br />
}<br />
}<br />
}<br />
return periods;<br />
}<br />
});<br />
<br />
/* jQuery extend now ignores nulls!<br />
@param target (object) the object to update<br />
@param props (object) the new settings<br />
@return (object) the updated object */<br />
function extendRemove(target, props) {<br />
$.extend(target, props);<br />
for (var name in props) {<br />
if (props[name] == null) {<br />
target[name] = null;<br />
}<br />
}<br />
return target;<br />
}<br />
<br />
/* Process the countdown functionality for a jQuery selection.<br />
@param command (string) the command to run (optional, default 'attach')<br />
@param options (object) the new settings to use for these countdown instances<br />
@return (jQuery) for chaining further calls */<br />
$.fn.countdown = function(options) {<br />
var otherArgs = Array.prototype.slice.call(arguments, 1);<br />
if (options == 'getTimes' || options == 'settings') {<br />
return $.countdown['_' + options + 'Countdown'].<br />
apply($.countdown, [this[0]].concat(otherArgs));<br />
}<br />
return this.each(function() {<br />
if (typeof options == 'string') {<br />
$.countdown['_' + options + 'Countdown'].apply($.countdown, [this].concat(otherArgs));<br />
}<br />
else {<br />
$.countdown._attachCountdown(this, options);<br />
}<br />
});<br />
};<br />
<br />
/* Initialise the countdown functionality. */<br />
$.countdown = new Countdown(); // singleton instance<br />
<br />
})(jQuery);<br />
<br />
<br />
</script><br />
<script type="text/javascript"><br />
$(function () {<br />
var jamboreeDay = new Date();<br />
jamboreeDay = new Date(2012, 10 - 1, 5);<br />
$('#jamboreeCountdown').countdown({<br />
until: jamboreeDay,<br />
/*onExpiry: liftOff*/<br />
compact: false,<br />
<br />
});<br />
});<br />
/*function liftOff() { <br />
alert('We are rolling!'); <br />
}*/<br />
</script><br />
<br />
<!-- Navigation scroll follow --><br />
<script type="text/javascript"><br />
$(window).scroll(function () { <br />
var scrollPos = $(window).scrollTop();<br />
if (scrollPos > 150) {<br />
$(".navbar").addClass("navbar-fixed-top");<br />
$(".navbar").removeClass("navbar-below-header");<br />
} else {<br />
$(".navbar").removeClass("navbar-fixed-top");<br />
$(".navbar").addClass("navbar-below-header");<br />
}<br />
if (scrollPos > 180) {<br />
$(".toc").addClass("stickBelowNavigation");<br />
} else {<br />
$(".toc").removeClass("stickBelowNavigation");<br />
}<br />
});<br />
</script><br />
<br />
<!-- iGem wiki hacks --><br />
<br />
<!-- Remove all empty <p> tags --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
$("p").filter( function() {<br />
return $.trim($(this).html()) == '';<br />
}).remove();<br />
});<br />
</script><br />
<br />
<!-- Remove "team" from the menubar --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
$("menubar > ul > li:last-child").remove();<br />
});<br />
</script><br />
<br />
<!-- Empty heading? - Then remove it.. --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
if ("" == "Vision") {<br />
$("#heading").remove();<br />
}<br />
});<br />
</script><br />
<br />
<!-- Fill the screen of index page on tablets --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
if ( $(window).width() < 1100 ) {<br />
$('#index-collapse').addClass('span12').removeClass('span9');<br />
}<br />
else {<br />
$('#index-collapse').removeClass('span12').addClass('span9');<br />
}<br />
});<br />
</script><br />
<br />
</body><br />
</html></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/FooterTeam:NTNU Trondheim/Templates/Footer2012-09-26T22:22:20Z<p>Oyas: </p>
<hr />
<div><html><br />
</div><!-- /container --><br />
<br />
<br />
<!-- Footer<br />
================================================== --><br />
<br />
<footer class="footer"><br />
<div class="container"><br />
<p class="pull-right top-pointer"><a href="#top"><i class="icon-arrow-up"></i></a></p><br />
<p>Designed and built with <a href="http://www.mediawiki.org/wiki/MediaWiki" target="_blank">MediaWiki</a>, <a href="http://jquery.com/" target="_blank">jQuery</a> and <a href="http://twitter.github.com/bootstrap/" target="_blank">Twitter Bootstrap</a>.</p><br />
<p>Icons by <a href="http://http://fortawesome.github.com/Font-Awesome/" target="_blank">Font Awesome</a>.</p><br />
<p><br /><br />
<i class="icon-envelope-alt"></i><a href="mailto:igem.ntnu@gmail.com"> igem.ntnu@gmail.com</a></p><br />
</div> <!-- /container --><br />
</footer><br />
<br />
<br />
<br />
<!-- Javascript<br />
================================================== --><br />
<!-- Placed at the end of the document so the pages load faster --><br />
<br />
<!-- Bootstrap --><br />
<script type="text/javascript" src="https://ajax.googleapis.com/ajax/libs/jquery/1.7.2/jquery.min.js"></script><br />
<script src="//netdna.bootstrapcdn.com/twitter-bootstrap/2.1.1/js/bootstrap.min.js"></script><br />
<br />
<br />
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<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-transition.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-alert.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-modal.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-dropdown.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-scrollspy.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-tab.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-tooltip.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-popover.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-button.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-collapse.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-carousel.js"></script><br />
<script src="http://folk.ntnu.no/oyas/igem/bootstrap/js/bootstrap-typeahead.js"></script>--><br />
<br />
<script type="text/javascript"><br />
$('.dropdown-toggle').dropdown()<br />
$('.carousel').carousel()<br />
</script><br />
<br />
<!-- Smooth scroll to top --><br />
<script type="text/javascript"><br />
$("a[href='#top']").click(function() {<br />
$("html, body").animate({ scrollTop: 0 }, "medium");<br />
return false;<br />
});<br />
</script><br />
<br />
<!-- jQuery countdown --><br />
<script type="text/javascript" src="http://folk.ntnu.no/oyas/igem/extras/js/jquery.countdown.js"></script><br />
<script type="text/javascript"><br />
$(function () {<br />
var jamboreeDay = new Date();<br />
jamboreeDay = new Date(2012, 10 - 1, 5);<br />
$('#jamboreeCountdown').countdown({<br />
until: jamboreeDay,<br />
/*onExpiry: liftOff*/<br />
compact: false,<br />
<br />
});<br />
});<br />
/*function liftOff() { <br />
alert('We are rolling!'); <br />
}*/<br />
</script><br />
<br />
<!-- Navigation scroll follow --><br />
<script type="text/javascript"><br />
$(window).scroll(function () { <br />
var scrollPos = $(window).scrollTop();<br />
if (scrollPos > 150) {<br />
$(".navbar").addClass("navbar-fixed-top");<br />
$(".navbar").removeClass("navbar-below-header");<br />
} else {<br />
$(".navbar").removeClass("navbar-fixed-top");<br />
$(".navbar").addClass("navbar-below-header");<br />
}<br />
if (scrollPos > 180) {<br />
$(".toc").addClass("stickBelowNavigation");<br />
} else {<br />
$(".toc").removeClass("stickBelowNavigation");<br />
}<br />
});<br />
</script><br />
<br />
<!-- iGem wiki hacks --><br />
<br />
<!-- Remove all empty <p> tags --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
$("p").filter( function() {<br />
return $.trim($(this).html()) == '';<br />
}).remove();<br />
});<br />
</script><br />
<br />
<!-- Remove "team" from the menubar --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
$("menubar > ul > li:last-child").remove();<br />
});<br />
</script><br />
<br />
<!-- Empty heading? - Then remove it.. --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
if ("" == "Vision") {<br />
$("#heading").remove();<br />
}<br />
});<br />
</script><br />
<br />
<!-- Fill the screen of index page on tablets --><br />
<script type="text/javascript"><br />
$(document).ready(function() {<br />
if ( $(window).width() < 1100 ) {<br />
$('#index-collapse').addClass('span12').removeClass('span9');<br />
}<br />
else {<br />
$('#index-collapse').removeClass('span12').addClass('span9');<br />
}<br />
});<br />
</script><br />
<br />
</body><br />
</html></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2012-09-26T22:22:00Z<p>Oyas: </p>
<hr />
<div><html lang="en"><br />
</p></div></div></div></div><br />
<head><br />
<meta http-equiv="content-type" content="text/html; charset=utf-8"><br />
<meta charset="utf-8"><br />
<meta name="viewport" content="width=device-width, initial-scale=1.0"><br />
<br />
<!-- Google fonts --> <br />
<link href='http://fonts.googleapis.com/css?family=Ubuntu:400,400italic,700,700italic' rel='stylesheet' type='text/css'><br />
<br />
<!-- Styles --><br />
<link href="http://folk.ntnu.no/oyas/igem3/bootstrap2/css/bootstrap.css" rel="stylesheet"><br />
<link href="http://folk.ntnu.no/oyas/igem3/extras/css/jquery.countdown.css" rel="stylesheet"><br />
<link href="http://folk.ntnu.no/oyas/igem3/extras/css/extras.css" rel="stylesheet"><br />
<br />
<!-- Responsive css must be added after top padding --><br />
<link href="http://folk.ntnu.no/oyas/igem3/bootstrap2/css/responsive.css" rel="stylesheet"><br />
<br />
<!-- HTML5 shim, for IE6-8 support of HTML5 elements --><br />
<!--[if lt IE 9]><br />
<script src="http://html5shim.googlecode.com/svn/trunk/html5.js"></script><br />
<![endif]--><br />
<!--[if lt IE 7]><br />
<script src="http://ie7-js.googlecode.com/svn/version/2.1(beta4)/IE7.js"></script><br />
<![endif]--><br />
<br />
</head><br />
<br />
<body><br />
<br />
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================================================== --><br />
<div id="fb-root"></div><br />
<script><br />
(function(d, s, id) {<br />
var js, fjs = d.getElementsByTagName(s)[0];<br />
if (d.getElementById(id)) return;<br />
js = d.createElement(s); js.id = id;<br />
js.src = "//connect.facebook.net/en_US/all.js#xfbml=1";<br />
fjs.parentNode.insertBefore(js, fjs);<br />
}(document, 'script', 'facebook-jssdk'));<br />
</script><br />
<br />
<br />
<!-- Header<br />
================================================== --><br />
<div class="header"><br />
<div class="container"><br />
<div class="heading visible-tablet visible-desktop"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
<div class="heading heading-small visible-phone"><a href="https://2012.igem.org/Team:NTNU_Trondheim">Search and Destroy</a></div><br />
<div class="sub-heading visible-tablet visible-desktop" id="sub-heading1"><br />
<span class="sub-heading-small">NTNU IS</span> <span style="font-size: 30pt; font-style: italic;">B.A.C.K.</span><br />
</div><br />
<div class="sub-heading visible-desktop" id="sub-heading2"><br />
B<span class="sub-heading-small">acterial</span> A<span class="sub-heading-small">nti</span>-C<span class="sub-heading-small">ancer</span>-K<span class="sub-heading-small">amikaze</span><br />
</div><br />
<div class="pull-right visible-desktop" style="text-align:right;" id="ntnu-logo"><br />
<a href="http://ntnu.edu" target="_blank"><img src="https://static.igem.org/mediawiki/2012/3/36/Ntnu_logo_en_scaled.png"/></a><br/><br />
<a href="https://2012.igem.org/Main_Page" target="_blank"><img id="igem-logo" src="https://static.igem.org/mediawiki/2012/c/cc/Igem_logo_scaled.png" /></a><br />
</div><br />
</div><br />
</div><br />
<br />
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================================================== --><br />
<div class="navbar navbar-below-header"><br />
<div class="navbar-inner"><br />
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<span class="icon-bar"></span><br />
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</a><br />
<div class="nav-collapse"><br />
<ul class="nav"><br />
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</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/HeaderTeam:NTNU Trondheim/Templates/Header2012-09-26T22:20:33Z<p>Oyas: </p>
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</div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/Templates/FooterTeam:NTNU Trondheim/Templates/Footer2012-09-26T22:18:28Z<p>Oyas: </p>
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<br />
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================================================== --><br />
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</html></div>Oyashttp://2012.igem.org/File:Igem_header_bacteria.pngFile:Igem header bacteria.png2012-09-26T22:04:39Z<p>Oyas: </p>
<hr />
<div></div>Oyashttp://2012.igem.org/Team:NTNU_Trondheim/OutreachTeam:NTNU Trondheim/Outreach2012-09-26T22:03:12Z<p>Oyas: </p>
<hr />
<div>{{:Team:NTNU_Trondheim/Templates/Header}}<br />
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<h1>Human practices <small>Bringing synthetic biology to the masses</small></h1><br />
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<br />
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<div class="container main-container"><br />
</html><br />
__TOC__<br />
==Human practices in summary==<br />
<br />
Our main human practices project will be to participate in Researchers' Night, which is an event for high school students and students from non-degree granting colleges. The goal of researchers' night is to show the students that research is fun, to inspire them, and to motivate them to take higher education.<br />
As a second outreach project, the team will be writing a chapter on different biobrick assembly methods in a textbook. <br />
<br />
We also concider our Matchmaker an important part of our outreach effort. Read more about it [https://2012.igem.org/wiki/index.php?title=Team:NTNU_Trondheim/Collaboration&action=submit#The_iGEM_Matchmaker here].<br />
<br />
==Researchers' Night==<br />
<br />
This year, the NTNU iGEM team is participating in Researchers' Night, which is an arrangement for high school students. This is the eighth year Researchers' night is being arranged, and it has traditionally been very popular at NTNU. Last year, over 1200 students visited the arrangement. This year, Researchers' night will be arranged the 28th of september, and we have been inveted to participate. We are really looking forward to it, since this is a unique opportunity to tell students about the possibilities of synthetic biology, and motivate them for a career in biotechnology. It seems that the students are looking forward to it as well, since this year's arrangement was fully booked in 4 minutes! Some photos from last year's event (taken by Kristina Jones, NTNU) can be seen in the carousel below.<br />
<br />
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<br />
This year, the 1100 participating students can visit 31 different stands, one of them being ours. To get the students to understand the concept of giving organisms new characteristic properties by putting together biobricks, we have made a biobrick construction kit, including both DNA and restriction enzymes (a picture of the DNA from the construction kit is given below). We hope that this construction kit will make it easier both to understand why we are able to put together biobricks using certain combinations of restriction enzymes, and we also hope we can teach them what the different sequences that makes a gene are used for.<br />
<br />
[[File:BiobrikkeByggesett.png|400px|thumb|center|Our BioBrick construction kit]]<br />
<br />
==Text book chapter on biobrick assembly methods==<br />
<br />
Early in the semester, we were asked by our advisor, Rahmi Lale, to write a chapter on biobrick assembly methods for a text book he and Svein Valla, Professor at Dept. of Biotechnology, NTNU, are editing. The text book in question is called 'DNA cloning methods', and is part of the book series '[http://www.springer.com/series/7651 Methods in Molecular Biology]' published by Humana Press. We will finalise the chapter after the Europe Regional Jamboree.<br />
<br />
<br />
{{:Team:NTNU_Trondheim/Templates/Sponsors}}<br />
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{{:Team:NTNU_Trondheim/Templates/Footer}}</div>Oyashttp://2012.igem.org/File:RN7.PNGFile:RN7.PNG2012-09-26T22:02:53Z<p>Oyas: </p>
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<div></div>Oyashttp://2012.igem.org/File:RN6.PNGFile:RN6.PNG2012-09-26T22:02:48Z<p>Oyas: </p>
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<div></div>Oyashttp://2012.igem.org/File:RN5.PNGFile:RN5.PNG2012-09-26T22:02:39Z<p>Oyas: </p>
<hr />
<div></div>Oyas