http://2008.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Romain.rousseau&year=&month=2008.igem.org - User contributions [en]2024-03-29T01:53:26ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:57:36Z<p>Romain.rousseau: </p>
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<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a alt="Team" title="Discover our team !" style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
<br><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project !"><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center><html><a title="Discover our team !" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
<br />
<br />
[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
</center><br />
<br><br />
{{Paris/Header|We are extremely grateful to the organizations that support our project:}}<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
<br />
Centre de Recherches Interdisciplinaires<br />
<br />
[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
<br />
|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
<br />
Paris Centre universités<br />
<br />
[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Embassy.png|250px]]<br />
<br />
Embassy of France in the United States<br />
<br />
[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
<br />
[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
<br />
Ibisc Laboratory<br />
<br />
[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
<hr /><br />
<center><br />
<html><br />
<a href="http://www4.clustrmaps.com/counter/maps.php?url=https://2008.igem.org/Team:Paris" id="clustrMapsLink"><br />
<img src="http://www4.clustrmaps.com/counter/index2.php?url=https://2008.igem.org/Team:Paris" border="0" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
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[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:57:00Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a alt="Team" title="Discover our team !" style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
<br><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br><br><br />
<br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project !"><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center><html><a title="Discover our team !" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
<br />
<br />
[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
</center><br />
<br><br />
{{Paris/Header|We are extremely grateful to the organizations that support our project:}}<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
<br />
Centre de Recherches Interdisciplinaires<br />
<br />
[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
<br />
|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
<br />
Paris Centre universités<br />
<br />
[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Embassy.png|250px]]<br />
<br />
Embassy of France in the United States<br />
<br />
[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
<br />
[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
<br />
Ibisc Laboratory<br />
<br />
[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
<hr /><br />
<center><br />
<html><br />
<a href="http://www4.clustrmaps.com/counter/maps.php?url=https://2008.igem.org/Team:Paris" id="clustrMapsLink"><br />
<img src="http://www4.clustrmaps.com/counter/index2.php?url=https://2008.igem.org/Team:Paris" border="0" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
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<br><br />
[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:55:11Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a alt="Team" title="Discover our team !" style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
<br><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br><br><br />
<br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project !"><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center><html><a title="Discover our team !" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
<br />
<br />
[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
<br><br />
{{Paris/Header|We are extremely grateful to the organizations that support our project:}}<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
<br />
Centre de Recherches Interdisciplinaires<br />
<br />
[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
<br />
|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
<br />
Paris Centre universités<br />
<br />
[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Embassy.png|250px]]<br />
<br />
Embassy of France in the United States<br />
<br />
[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
<br />
[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
<br />
Ibisc Laboratory<br />
<br />
[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
<hr /><br />
<html><br />
<a href="http://www4.clustrmaps.com/counter/maps.php?url=https://2008.igem.org/Team:Paris" id="clustrMapsLink"><br />
<img src="http://www4.clustrmaps.com/counter/index2.php?url=https://2008.igem.org/Team:Paris" border="0" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
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<br><br />
[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:54:05Z<p>Romain.rousseau: /* We are extremely grateful to the organizations that support our project: */</p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a alt="Team" title="Discover our team !" style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
</center><br />
<br><br />
<br><br />
<br />
<center><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br><br><br />
<br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project !"><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center><html><a title="Discover our team !" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
<br />
<br />
[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
</center><br />
<br><br />
{{Paris/Header|We are extremely grateful to the organizations that support our project:}}<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
<br />
Centre de Recherches Interdisciplinaires<br />
<br />
[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
<br />
|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
<br />
Paris Centre universités<br />
<br />
[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Embassy.png|250px]]<br />
<br />
Embassy of France in the United States<br />
<br />
[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
<br />
[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
<br />
Ibisc Laboratory<br />
<br />
[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
<hr /><br />
<html><br />
<a href="http://www4.clustrmaps.com/counter/maps.php?url=https://2008.igem.org/Team:Paris" id="clustrMapsLink"><br />
<img src="http://www4.clustrmaps.com/counter/index2.php?url=https://2008.igem.org/Team:Paris" border="0" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
<br /><br />
<a href="http://www.easycounter.com/"><br />
<img src="http://www.easycounter.com/counter.php?bloup" border="0" /><br />
</a><br />
</html><br />
<br><br />
[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:49:30Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a alt="Team" title="Discover our team !" style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
</center><br />
<br><br />
<br><br />
<br />
<center><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br><br><br />
<br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project !"><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center><html><a title="Discover our team !" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
<br />
<br />
[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
</center><br />
<br><br />
=We are extremely grateful to the organizations that support our project: =<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
<br />
Centre de Recherches Interdisciplinaires<br />
<br />
[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
<br />
|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
<br />
Paris Centre universités<br />
<br />
[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Embassy.png|250px]]<br />
<br />
Embassy of France in the United States<br />
<br />
[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
<br />
[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
<br />
Ibisc Laboratory<br />
<br />
[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
<hr /><br />
<center><html><br />
<a href="http://www4.clustrmaps.com/counter/maps.php?url=https://2008.igem.org/Team:Paris" id="clustrMapsLink"><br />
<img src="http://www4.clustrmaps.com/counter/index2.php?url=https://2008.igem.org/Team:Paris" border="0" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
<br /><br />
<a href="http://www.easycounter.com/"><br />
<img src="http://www.easycounter.com/counter.php?bloup" border="0" /><br />
</a><br />
</html><br />
<br><br />
[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:49:05Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a alt="Team" title="Discover our team ! " style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
</center><br />
<br><br />
<br><br />
<br />
<center><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br><br><br />
<br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project ! "><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center><html><a title="Discover our team ! " href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
<br />
<br />
[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
</center><br />
<br><br />
=We are extremely grateful to the organizations that support our project: =<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
<br />
Centre de Recherches Interdisciplinaires<br />
<br />
[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
<br />
|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
<br />
Paris Centre universités<br />
<br />
[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Embassy.png|250px]]<br />
<br />
Embassy of France in the United States<br />
<br />
[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
<br />
[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
<br />
Ibisc Laboratory<br />
<br />
[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
<hr /><br />
<center><html><br />
<a href="http://www4.clustrmaps.com/counter/maps.php?url=https://2008.igem.org/Team:Paris" id="clustrMapsLink"><br />
<img src="http://www4.clustrmaps.com/counter/index2.php?url=https://2008.igem.org/Team:Paris" border="0" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
<br /><br />
<a href="http://www.easycounter.com/"><br />
<img src="http://www.easycounter.com/counter.php?bloup" border="0" /><br />
</a><br />
</html><br />
<br><br />
[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:46:47Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
</center><br />
<br><br />
<br><br />
<br />
<center><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br><br><br />
<br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project !"><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center>[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
<html><a title="Discover our team!" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
</center><br />
<br><br />
<br><br />
<br><br />
<br><br />
=We are extremely grateful to the organizations that support our project=<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
<br />
Centre de Recherches Interdisciplinaires<br />
<br />
[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
<br />
|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
<br />
Paris Centre universités<br />
<br />
[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Embassy.png|250px]]<br />
<br />
Embassy of France in the United States<br />
<br />
[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
<br />
[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
<br />
Ibisc Laboratory<br />
<br />
[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
<hr /><br />
<center><html><br />
<a href="http://www4.clustrmaps.com/counter/maps.php?url=https://2008.igem.org/Team:Paris" id="clustrMapsLink"><br />
<img src="http://www4.clustrmaps.com/counter/index2.php?url=https://2008.igem.org/Team:Paris" border="0" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
<br /><br />
<a href="http://www.easycounter.com/"><br />
<img src="http://www.easycounter.com/counter.php?bloup" border="0" /><br />
</a><br />
</html><br />
<br><br />
[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:45:15Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
<br />
<html><div style="text-align:justify; background: url(https://static.igem.org/mediawiki/2008/f/fc/IGEM_PARIS_2008_fond4.jpg); background-repeat:no-repeat; margin=0;"></html><br />
<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
<br/><br />
<div style="font-size: 2em;">by the <html><a style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
</center><br />
<br><br />
<br><br />
<br />
<center><br />
<html><br />
<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
</html><br />
</center><br />
<br />
<br><br />
<br><br />
Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
<br><br><br><br />
<br />
<center><html><a href="https://2008.igem.org/Team:Paris/Project" alt="Project" title="Learn more about our project!"><img width=350px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
<br />
<br />
In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
<br />
<br />
<br />
<center>[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
<html><a title="Discover our team!" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
</center><br />
<br><br />
<br><br />
<br><br />
<br><br />
=We are extremely grateful to the organizations that support our project=<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
<br />
Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
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|width=25%| [[Image:Logocri.jpg|115px]]<br />
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Centre de Recherches Interdisciplinaires<br />
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[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
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|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
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Paris Centre universités<br />
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[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
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|-<br />
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|-<br />
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|[[Image:Embassy.png|250px]]<br />
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Embassy of France in the United States<br />
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[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
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|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
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[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
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|[[Image:Qiagen.GIF|60px]]<br />
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Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
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|[[Image:Ibisc.jpeg|100px]]<br />
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Ibisc Laboratory<br />
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[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
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|[[Image:Gdrbim.jpg|100px]]<br />
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GdR BiM<br />
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[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
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|[[Image:Logo INRIA.jpg|180px]]<br />
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INRIA<br />
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[http://www.inria.fr/ www.inria.fr]<br />
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|}<br />
<hr /><br />
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[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:ParisTeam:Paris2008-10-30T07:44:26Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
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<br><br />
<center><html><div style="color:#275D96; font-size:4em;">The BacteriO'Clock</div></html><br />
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<div style="font-size: 2em;">by the <html><a style="text-decoration: underline;" href="https://2008.igem.org/Team:Paris/Team">Paris iGEM Team</a></html></div><br />
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<embed width="320" height="240" src="http://biosynthetique.free.fr/videos/flv_player/flvplayer.swf?autostart=true&amp;repeat&amp;file=http://perso.strepsiade.org/~gvieira/out2.flv" quality="high" type="application/x-shockwave-flash" allowfullscreen="true" /><br />
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Have you ever dreamed of a new way of thinking everyday life objects? Be sure, this could one day become true, under the form of our '''BacteriO'Clock !''' A simple test tube containing our modified bacteria might give you the time, directly from living organisms, the hours of the day being '''color-coded''', and '''oscillations''' ensuring the repeated periodic behavior. Inspired by the flagellar motor machine, our clock at this stage of developpement serves as a '''chassis''' to be used in building future modular molecular machines in an optimized and coordinated manner. It may serve as well to control in an optimized fashion '''multi-reaction pathways'''.<br />
<br />
<br><br />
To achieve this incredible project, we relied on a well characterized genetic structure that allows a specific sequence of fluorescent proteins expression to occur in a First In - First Out order. This '''FIFO''' behavior enables us to define a rich color encoding of day time. An additional negative feedback loop generates clock oscillations! <br />
<br><br><br />
Based on in-depth studies and experimentally measured parameters we developed predictive models that suggest that this core system is not likely to demonstrate stable oscillations due to damping phenomenon leading to a stationary steady-state. Importantly, our modeling approach provided us with alternative improved designs that should lead in principle to an optimized '''BacteriO'Clock'''. In particular, this is achieved through '''synchronization at the population level''' by adopting a quorum sensing feedback mediated by HSL production that enforces a necessary delay and provides an elegant cellular synchronization mechanism : <br />
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<center><html><a href="https://2008.igem.org/Team:Paris/Project"><img width=500px src="https://static.igem.org/mediawiki/2008/5/58/Unimo.png" /></a></html></center><br />
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In a nutshell, the new trendy item which will make every biologist in your lab jealous will soon be available. Through a long-cloning period, '''we now have all biobricks needed and much of the final constructions'''. Furthermore, we initiated an extensive characterization plan of these parts. If you manage to wait for the updates, you might even get the version that automatically reacts to daylight saving time ;)<br />
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<center>[[Team:Paris/Notebook/Freezer|Freezer]] || [[Team:Paris/Notebook|Notebook]]<br />
<html><a title="Discover our team!" href="https://2008.igem.org/Team:Paris/Team"><img style="padding: 5px; border: orange dashed 1px;" src="https://static.igem.org/mediawiki/2008/1/19/ParisTeam.jpg" width="300px" /></a></html><br />
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=We are extremely grateful to the organizations that support our project=<br />
{|<br />
|width=25%| [[Image:bettencourt.png]]<br />
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Bettencourt-Schueller Foundation<br />
<br />
[http://www.fondationbs.org/ www.fondationbs.org]<br />
<br />
|width=25%| [[Image:Logocri.jpg|115px]]<br />
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Centre de Recherches Interdisciplinaires<br />
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[http://www.cri-paris.org/fr/cri/ www.cri-paris.org/fr/cri/]<br />
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|width=25%| [[Image:LogoPCU1.jpg|100px]]<br />
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Paris Centre universités<br />
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[http://www.pariscentreuniversites.fr/ www.pariscentreuniversites.fr]<br />
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|-<br />
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|-<br />
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|[[Image:Embassy.png|250px]]<br />
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Embassy of France in the United States<br />
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[http://www.ambafrance-us.org/ www.ambafrance-us.org]<br />
<br />
|[[Image:FacMed.png|140px]]<br />
Faculté de Médecine Université Paris Descartes<br />
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[http://www.medecine.univ-paris5.fr/ www.medecine.univ-paris5.fr]<br />
<br />
|[[Image:Qiagen.GIF|60px]]<br />
<br />
Qiagen<br />
<br />
[http://www.qiagen.com/ www.qiagen.com/]<br />
|-<br />
|-<br />
|-<br />
<br />
|[[Image:Ibisc.jpeg|100px]]<br />
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Ibisc Laboratory<br />
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[http://www.ibisc.univ-evry.fr/ www.ibisc.univ-evry.fr]<br />
<br />
<br />
|[[Image:Gdrbim.jpg|100px]]<br />
<br />
GdR BiM<br />
<br />
[http://www.gdr-bim.u-psud.fr/ www.gdr-bim.u-psud.fr]<br />
<br />
|[[Image:Logo INRIA.jpg|180px]]<br />
<br />
INRIA<br />
<br />
[http://www.inria.fr/ www.inria.fr]<br />
<br />
|}<br />
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[[Team:Paris/Museum|Have fun visiting our old wiki!]]</center></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/ConstructionTeam:Paris/Construction2008-10-30T07:31:53Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Model constructions: from the modelling to the characterization}}<br />
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'''Have a look at our [[Team:Paris/Notebook|notebook]].'''<br />
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<br />
Our project BacterioClock is based on an oscillating FIFO synchronized at the population level. To obtain and have preliminary results of this system we divided it in three "little" modules as the modeling team exposed them previously.<br />
=The First is to demonstrate the [[Team:Paris/Analysis#FIFO_behaviour|FIFO]] which constitutes our core system=<br />
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A simple manner is to implement two class II promoter followed by fluorescent reporter genes like GFP in a bacteria strain that is able to produce the flagella. <br />
<br />
By this way, we are able to see in single cell the order of the activation of pFliL then pFlgA then pFlhB and then the inactivation in the same order as a FIFO will do.<br />
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These constructions are equivalent to the experiments realized by Uri Alon in the article "Using quantitative Blueprint to reprogram the Dynamics of the Flagella Gene Network" Kalir S, Alon U. Cell 2004.<br />
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A better way to observe the FIFO is to study the system with an inducible regulator of class II gene in a bacteria strain deleted for this gene. We are extremely grateful for Alon U. that send us the inducible gene ''FlhDC'', and ''FliA'' in pBad18 plasmid.<br />
These plasmids with our own araC/pBad-EnvZ* will allowed us to study the system in mutated strains that we have in our lab (ΔFlhD, ΔFliA, ΔFlgM, ΔEnvZ). By cotransforming in mutated strains the inducible regulators of the class II promoters with one of this promoter associated to a fluorescent protein, we could characterize the influence of the master regulators of the flagella on their promoter. The fluorescence is normalized to the OD<sub>600</sub>.<br />
<br>For example below are some constructions to see and induce the FIFO:<br />
<br><br><br><br />
[[Image:Felipe01.jpg|500px|center|thumb|pFliL alone]]<br><br><br><br />
[[Image:Felipe02.jpg|500px|center|thumb|3 promoters of the FIFO in one Cell]]<br><br><br><br />
[[Image:Felipe03.jpg|500px|center|thumb|FIFO and oscillating constructions in a single cell due to the flhDC repression by EnvZ*. ]]<br><br><br><br />
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<br />
To perform such a cotransformation we take care about the ORI of each low copy plasmid which are often incompatible and we made all our final constructions in the pSB4T5 plasmid that care the only one resistance that was not already used and the pSC101 ORI which is compatible with the ColE1.<br />
<br />
We could see the FIFO in a more convenient manner with the assembly of two class II promoters associated to different fluorophores. Then we could observed two kinds of fluorescents in One Cell along time. <br />
One problem that could occur is the interaction between the different fluorescents proteins due to the cross-over of their emission and excitation spectra and moreover in our final system that count three fluorophores.<br />
<br />
[[Image:Spectre.jpg |600px |thumb| emission and excitation spectra of ECFP, YFP and mRFP]]<br />
<br />
<br>The choice of ECFP, YFP and mRFP is based upon this consideration and upon the order of appearing in the FIFO.<br />
Indeed by putting the mRFP in last position, we improve the visualization of the FIFO like the maturation time of the protein is about of one hour.<br />
<br />
We thank a lot Arnau Montagu, advisor of the iGEM Valencia Team. He advised us, after his team had troubleshooting with fluorescence, to take care about ''spurious FRET''. This phenomenon occurs even with non covered fluorescent spectra when the concentration of two fluorophores is sufficient so they are enough closer to induce some FRET. But it could happen at lower concentration than 40 μM. <br><br />
<br>- "DsRed as a Potential FRET Partner with CFP and GFP" Michael G. Erickson, Daniel L. Moon, and David T. Yue Biophysical Journal 2003. <br />
<br>- "A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP<br />
Sensor as an Example" Gerard N. M. van der Krogt, Janneke Ogink, Bas Ponsioen, Kees Jalink PLoS 2008.<br />
<br />
<br><br />
<br />
=[[Team:Paris/Analysis#Oscillations|Oscillating]] module=<br />
Few approaches have been thought in order to create the oscillating module. The pTet/TetR system that allows us to precisely control the inhibition of TetR on the Tet promoter. Another system use the natural inhibition of OmpR and of EnvZ via OmpR. We finally perform this system that we have developed since we know thanks to the modeling team that the oscillations are the only way to see the full development of the FIFO.<br />
<br />
=Synchronization module=<br />
The modeling simulation showed us that to have some oscillations that are difficult to obtain in a simple feedforward loop without the delay introduce by the quorum sensing synchronization, and for us it is more interesting to have the FIFO behavior at the population level. We did not achieved this module but we started the construction of it and have already some intermediate parts that you will find below.<br />
<br />
=The constructions we were able to obtain were characterised by fluorescence microscopy (see examples below):=<br />
[[Image:ParisGfp.jpg|thumb|300px|center|Green fluorescence]]<br />
[[Image:ParisRfp.jpg|thumb|300px|center|Red Fluorescence]]<br />
[[Image:ParisCfp.jpg|thumb|300px|center|Cyan Fluorescence]]<br />
<br />
= Table of the constructions we realized=<br />
{|<br />
|- style="background: #649cd7;"<br />
!colspan=4 style="background: #649cd7" | FIFO<br />
|- style="background: #649cd7; text-align: center;"<br />
|width=30%| Construction<br />
|width=55%| Description<br />
|width=15%| Validation Date<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136039 pFliL(RBS WT)-ECFP tripart (LVA+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is ECFP fused with a LVA tag.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136037 pFliL(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFliL is GFP.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136012 pFlgA(+ RBS WT) - YFP Tripart (LVA+)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136013 pFlgA(+ RBS WT) - YFP Tripart (LVA-)]<br />
|align=|Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused without a LVA tag. <br />
|align="center"| [[Team:Paris/August 22|August 22]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136054 pFlgA(+ RBS WT)-YFP tripart (AAV+)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is EYFP fused with a AAV tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136053 pFlgA(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlgA is GFP. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136055 pFlhB(+ RBS WT)-mRFP tripart (+LVA)]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is mRFP with a LVA tag. <br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_reporter.png]]<br>[http://partsregistry.org/Part:BBa_K136065 pFlhB(+ RBS WT)-GFP]<br />
|align=| Natural Promoter of E. coli regulated by FlhDC and FliA. The natural rbs of the downstream gene is conserved for a more natural expression of the following gene. The fluorescent marker associated with pFlhB is GFP. <br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''OSCILLATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136064 pFliL(+ RBS WT)-EnvZ*]<br />
|align=| pFliL is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 26|October 26]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136069 pFliL(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFliL.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136051 pFlhB(+ RBS WT)-EnvZ*]<br />
|align=| pFlhB is regulated by FlhDC and EnvZ regulates pFlhDC thanks to a negative feedback loop, origin of an oscillation behavior.<br />
|align="center"| [[Team:Paris/October 24|October 24]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136070 pFlhB(+ RBS WT)-EnvZ*-GFP generator]<br />
|align=| The same construction as before but with a GFP in order to follow the dynamic of expression of pFlhB.<br />
|align="center"| [[Team:Paris/October 29|October 29]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136066 araC/pBad-EnvZ*]<br />
|align=| A construction made for characterizing the repression power of the mutated EnvZ on our system<br />
|align="center"| [[Team:Paris/October 27|October 27]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''SYNCHRONISATION'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136029 pLas - gfp Tripart]<br />
|align=| Construction for characterization of the pLas promoter<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136027 pLas - ECFP]<br />
|align=| The same as before but with another fluorophore<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136024 strongest promoter-rbs-lasR-dble ter]<br />
|align=| A construction for a strong constitutive expression of the Las Regulator in order to get maximal expression in presence of AHL<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_regulatory.png]][[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136023 Strong promoter-rbs-lasR-dble ter]<br />
|align=| A derived construction of the previous one for smaller constitutive expression of LasR in the Bacteria.<br />
|align="center"| [[Team:Paris/August 20|August 20]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136044 Strongest RBS (1)- LasR activator (+LVA)]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136048 rbs-lasR + dbl ter]<br />
|align=| Intermediary construction. We uses different rbs for Las R in order to get various response dynamic to the presence of AHL<br />
|align="center"| [[Team:Paris/August 19|August 19]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136052 lasR activator + LVA - Double Terminator]<br />
|align=| Intermediary construction<br />
|align="center"| [[Team:Paris/August 1|August 1]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br>[http://partsregistry.org/Part:BBa_K136057 RBS lasI - GFP tripart]<br />
|align=| Construction that has to be associated with the FIFO. We will know when LasI is expressed and then when AHL is produced.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_rbs.png]][[Image:Icon_coding.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]<br> [http://partsregistry.org/Part:BBa_K136056 RBS-lasI - ECFP]<br />
|align=| The same as the previous one but with another fluorophore.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|-<br />
|colspan=4 style="background: #649cd7" | <center>'''NEW GENE AND PROMOTER BIOBRICKS'''</center><br />
|-<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136010 pFliA]<br />
|align=| Natural promoter of E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC.<br />
|align="center"| [[Team:Paris/October 04|October 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136009 pFliL (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136006 pFlgA (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA..<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br> pFlgB (+RBS WT)<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Part_icon_regulatory.png]]<br>[http://partsregistry.org/Part:BBa_K136008 pFlhB (+RBS WT)]<br />
|align=| Natural promoter with the wild type rbs of its associated gene in E. coli, extracted by PCR and then inserted in a biobrick vector. pFiA is regulated by FlhDC and FliA.<br />
|align="center"| [[Team:Paris/October 10|October 10]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>[http://partsregistry.org/Part:BBa_K136046 EnvZ*]<br />
|align=| EnvZ is a membrane protein involved into the osmotic response and is interacting with OmpR that will bind the promoter of FlhDC ad will repress it. EnvZ* is a mutated form of the coding sequence, giving a phenotype where pFlhDC is completely repressed. EnvZ has been extracted by PVR for the correct mutant strain.<br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | [[Image:Icon_coding.png]]<br>FliA<br />
|align=| FliA is a sigma factor involved into the regulation of very various genes, the promoter used in the FIFO were selected to be higly responding to FliA. In order to have the FliA gene possibly used as biobrick, we mutated 2 sites (EcoRI and PstI) inside the FliA sequence by assemby PCR. <br />
|align="center"| [[Team:Paris/September 04|September 04]]<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" |[[Image:Icon_coding.png]]<br>FlhDC<br />
|align=| FlhDC is the main regulator of the flagella genetic system in E. coli. FlhDC regulates all the promoter of the FIFO.<br />
|align="center"| [[Team:Paris/August 13|August 13]]<br />
|}<br />
<br />
{{Paris/Navig|Team:Paris/Construction}}</div>Romain.rousseauhttp://2008.igem.org/Template:Paris/MenuTemplate:Paris/Menu2008-10-30T07:30:15Z<p>Romain.rousseau: </p>
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|style="background:#fff"|</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/SandboxTeam:Paris/Sandbox2008-10-30T07:29:44Z<p>Romain.rousseau: </p>
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__TOC__<br />
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<br />
=A=<br />
==Aa==<br />
==Ab==<br />
=B=<br />
=C=<br />
=D=<br />
==Da==<br />
==Db==<br />
===Db1===<br />
===Db2===<br />
===Db3===<br />
=E=</div>Romain.rousseauhttp://2008.igem.org/Template:Paris/MenuTemplate:Paris/Menu2008-10-30T07:27:19Z<p>Romain.rousseau: </p>
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{|cellspacing="5" cellpadding="10" style="margin-top: 3px; background:#649CD7; width: 965px;"<br />
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|style="background:#fff"|</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/SandboxTeam:Paris/Sandbox2008-10-30T07:25:43Z<p>Romain.rousseau: </p>
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http://css.alsacreations.com/Construction-de-menus-en-CSS/Faire-un-menu-avec-onglets-en-CSS-avec-before-after<br />
<br />
<div class="collapsed">__TOC__</div><br />
<br />
<br />
=A=<br />
==Aa==<br />
==Ab==<br />
=B=<br />
=C=<br />
=D=<br />
==Da==<br />
==Db==<br />
===Db1===<br />
===Db2===<br />
===Db3===<br />
=E=</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/SandboxTeam:Paris/Sandbox2008-10-30T07:16:55Z<p>Romain.rousseau: </p>
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height: 150px;<br />
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}<br />
<br />
#menu {<br />
background: url(https://static.igem.org/mediawiki/2008/5/58/Gradient.png);<br />
}<br />
<br />
#menu ul {<br />
margin: 0;<br />
padding: 0;<br />
list-style: none;<br />
text-align: center;<br />
}<br />
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display: inline;<br />
}<br />
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font: 2em "Trebuchet MS",Arial,sans-serif;<br />
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text-align: center;<br />
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*/<br />
}<br />
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color:white;<br />
background: blue;<br />
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}<br />
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background: url(https://static.igem.org/mediawiki/2008/4/41/Arrow.png) no-repeat;<br />
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<li><a href="https://2008.igem.org/Team:Paris/Analysis/page3">Page 3</a></li><br />
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<br />
http://css.alsacreations.com/Construction-de-menus-en-CSS/Faire-un-menu-avec-onglets-en-CSS-avec-before-after<br />
<br />
<div class="collapsed">__TOC__</div><br />
<br />
<br />
=A=<br />
==Aa==<br />
==Ab==<br />
=B=<br />
=C=<br />
=D=<br />
==Da==<br />
==Db==<br />
===Db1===<br />
===Db2===<br />
===Db3===<br />
=E=</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/SandboxTeam:Paris/Sandbox2008-10-30T07:16:44Z<p>Romain.rousseau: </p>
<hr />
<div><html><br />
<style type="text/css"><br />
<br />
#header {<br />
height: 150px;<br />
background: url(https://static.igem.org/mediawiki/2008/4/4c/Panoramique.jpg) no-repeat center;<br />
}<br />
<br />
#menu {<br />
background: url(https://static.igem.org/mediawiki/2008/5/58/Gradient.png);<br />
}<br />
<br />
#menu ul {<br />
margin: 0;<br />
padding: 0;<br />
list-style: none;<br />
text-align: center;<br />
}<br />
<br />
#menu li {<br />
display: inline;<br />
}<br />
<br />
#menu li a {<br />
padding: 4px 20px;<br />
font: 2em "Trebuchet MS",Arial,sans-serif;<br />
text-decoration: none;<br />
/*<br />
text-align: center;<br />
color: black;<br />
background: #c00;<br />
border: 1px solid #600;<br />
line-height: 1em;<br />
*/<br />
}<br />
<br />
#menu li a:hover, #menu li a:focus, #menu li a:active {<br />
color:white;<br />
background: blue;<br />
text-decoration: underline;<br />
}<br />
<br />
#menu a:after {<br />
background: url(https://static.igem.org/mediawiki/2008/4/41/Arrow.png) no-repeat;<br />
content: "";<br />
padding: 0 25px 0 0;<br />
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}<br />
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width: 0;<br />
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<li><a href="https://2008.igem.org/Team:Paris/Analysis/page3">Page 3</a></li><br />
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<br />
http://css.alsacreations.com/Construction-de-menus-en-CSS/Faire-un-menu-avec-onglets-en-CSS-avec-before-after<br />
<br />
<div class="collapse">__TOC__</div><br />
<br />
<br />
=A=<br />
==Aa==<br />
==Ab==<br />
=B=<br />
=C=<br />
=D=<br />
==Da==<br />
==Db==<br />
===Db1===<br />
===Db2===<br />
===Db3===<br />
=E=</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/SandboxTeam:Paris/Sandbox2008-10-30T07:16:23Z<p>Romain.rousseau: </p>
<hr />
<div><html><br />
<style type="text/css"><br />
<br />
#header {<br />
height: 150px;<br />
background: url(https://static.igem.org/mediawiki/2008/4/4c/Panoramique.jpg) no-repeat center;<br />
}<br />
<br />
#menu {<br />
background: url(https://static.igem.org/mediawiki/2008/5/58/Gradient.png);<br />
}<br />
<br />
#menu ul {<br />
margin: 0;<br />
padding: 0;<br />
list-style: none;<br />
text-align: center;<br />
}<br />
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#menu li {<br />
display: inline;<br />
}<br />
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padding: 4px 20px;<br />
font: 2em "Trebuchet MS",Arial,sans-serif;<br />
text-decoration: none;<br />
/*<br />
text-align: center;<br />
color: black;<br />
background: #c00;<br />
border: 1px solid #600;<br />
line-height: 1em;<br />
*/<br />
}<br />
<br />
#menu li a:hover, #menu li a:focus, #menu li a:active {<br />
color:white;<br />
background: blue;<br />
text-decoration: underline;<br />
}<br />
<br />
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background: url(https://static.igem.org/mediawiki/2008/4/41/Arrow.png) no-repeat;<br />
content: "";<br />
padding: 0 25px 0 0;<br />
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}<br />
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<li><a href="https://2008.igem.org/Team:Paris/Analysis/page1">Page 1</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/page2">Page 2</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/page3">Page 3</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/page4">Page 4</a></li><br />
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</html><br />
<br />
<br />
http://css.alsacreations.com/Construction-de-menus-en-CSS/Faire-un-menu-avec-onglets-en-CSS-avec-before-after<br />
<br />
__TOC__<br />
<br />
<br />
=A=<br />
==Aa==<br />
==Ab==<br />
=B=<br />
=C=<br />
=D=<br />
==Da==<br />
==Db==<br />
===Db1===<br />
===Db2===<br />
===Db3===<br />
=E=</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/SandboxTeam:Paris/Sandbox2008-10-30T07:15:36Z<p>Romain.rousseau: </p>
<hr />
<div><html><br />
<style type="text/css"><br />
<br />
#header {<br />
height: 150px;<br />
background: url(https://static.igem.org/mediawiki/2008/4/4c/Panoramique.jpg) no-repeat center;<br />
}<br />
<br />
#menu {<br />
background: url(https://static.igem.org/mediawiki/2008/5/58/Gradient.png);<br />
}<br />
<br />
#menu ul {<br />
margin: 0;<br />
padding: 0;<br />
list-style: none;<br />
text-align: center;<br />
}<br />
<br />
#menu li {<br />
display: inline;<br />
}<br />
<br />
#menu li a {<br />
padding: 4px 20px;<br />
font: 2em "Trebuchet MS",Arial,sans-serif;<br />
text-decoration: none;<br />
/*<br />
text-align: center;<br />
color: black;<br />
background: #c00;<br />
border: 1px solid #600;<br />
line-height: 1em;<br />
*/<br />
}<br />
<br />
#menu li a:hover, #menu li a:focus, #menu li a:active {<br />
color:white;<br />
background: blue;<br />
text-decoration: underline;<br />
}<br />
<br />
#menu a:after {<br />
background: url(https://static.igem.org/mediawiki/2008/4/41/Arrow.png) no-repeat;<br />
content: "";<br />
padding: 0 25px 0 0;<br />
/*height: 25px; width: 25px;*/<br />
}<br />
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#menu li:last-child a:after {<br />
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pre {font-size: 1.2em}<br />
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<div id="links"><br />
<ul><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/page1">Page 1</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/page2">Page 2</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/page3">Page 3</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/page4">Page 4</a></li><br />
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</html><br />
<br />
<br />
http://css.alsacreations.com/Construction-de-menus-en-CSS/Faire-un-menu-avec-onglets-en-CSS-avec-before-after<br />
<br />
__TOC__</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Modeling/estimationTeam:Paris/Modeling/estimation2008-10-30T07:07:55Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
<br />
=Parameters Estimation=<br />
<br />
==Getting a "complexation function" from convenient datas==<br />
Therefore, we have written a little module which can estimate the parameters of a [[Team:Paris/Modeling/Programs|complexation function]], even with some noise and few data available.<br />
<br />
The method we have employed is essentially based on a least-square optimization.<br />
<br />
==Getting convenient datas==<br />
Thus, we need experimental datas. To quantify the strength of a transcription factor on a promoter, we will use measurements of GFP fluorescence, and compare to the strength of the constitutive promoter [[http://partsregistry.org/Measurement/SPU/Learn J23101]], as it was proposed by the iGEM competition.<br />
The datas we are looking for must appear as a table of values, giving several "expression rates" with their corresponding "transcription factor concentrations".<br />
<br />
==First hypothesis==<br />
For this aim, we made several hypothesis, which we will verify as good as it is possible for us :<br />
<br />
'''(1)''' We do not take into acount the 'traduction' phase (see however [[Team:Paris/Modeling/estimation#The_Problem_of_the_RBS|considerations on RBS]]), so we directly correlate the transcription of a gene with the concentration of its protein.<br />
<br />
'''(2)''' We assume that, whatever is the gene behind the promoter, its expression depends only of the transcription factor of the promoter, and not, for instance, of the weight of this gene. That's why comparing promoter strength is relevent only if the genes behind have similar length.<br />
<br />
'''(3)''' We consider that the activity of a promoter is well described as a a '''transduction rate''' ( ''' ''b'' ''', we will later note ''' ''&beta;'' ''' = ''b*p<sub>tot</sub>'', where ''p<sub>tot</sub>'' denotes the total number of promoters ''p'' present in the cell), multiplied by the amount of '''efficient promoters''' (''p<sub>eff</sub>''), depending on the quantity of its '''transcription factor''' (''TF''), and on the coefficients of their [[Team:Paris/Modeling/Programs#Complexations_Caracterisation|complexation reaction]] (''' ''K'' ''' et ''' ''n'' '''), typically leading to a [[Team:Paris/Modeling/Programs#Hill_Functions|Hill function]].<br />
Thus, we suppose that the protein concentration (''Prot'') follows this equation :<br />
<center> [[Image:dProt.jpg|center]] </center><br />
where ''&gamma;'' is a constant, due to degradation and of dilution of the protein, along time and cell divisions.<br />
Therefore, if we consider a '''steady-state''', for given concentration of the transcription factor, we will have : <center> [[Image:steadystate.jpg|center]] </center><br />
<br />
'''(4)''' Endly, knowing ''&gamma;'' will give us the data we are looking for. In a first approach, we assume that, as long as the barcteria are in their '''phase of exponential growth''', the degradation is far smaller than the dilution, and can be omitted. But we will probably discuss that later (see [[Team:Paris/Modeling/estimation#The_Problem_of_the_RBS|considerations on RBS]]).<br />
First, we wanted to keep a permanent exponential phase of growth with the system of Ron Weiss (see [[Team:Paris/Project|our project]]), but we finally (to simplify our already huge construction!) decided to put the cells-culture in a '''chemostat''' (see [[Team:Paris/Modeling/hill_approach#Bio-Chemical_General_Assumptions|Bio-Chemical General Assumptions]]).<br />
<br />
'''(5)''' Unless we find further documents dealing with the relation between the intensity of fluorescence and the concentration of GFP, we will directly use the measure in fluorescence, that we will treat as a protein concentration, more or less ''arbitrary normalised''.<br />
<br>Actually, we will use the linear relation between GFP mut3B concentration (nM) and fluorescence (au), given in the registry [[Team:Paris/Modeling/Bibliography|[6]]]. The conversion factor is 79,429.<br />
<br />
<br />
==RBS Issue==<br />
The promoter is not the only one factor which control the expression of the protein... in particular, the ''traduction phenomenon'' is almost as important as the ''transduction''. As we decided not to take into acount the traduction, that means that we do not want to deal with mRNA and ribosomes ; nevertheless, as we aimed in the "APE approach" to simulate as precisely as possible the involved concentrations, we must ''integrate in the traduction simulation the most important influences of the transduction''.<br />
<br />
Therefore, rigorously, we must describe an induction (or repression) by the association "promoter + specific RBS".<br />
<br />
Still, as it is proposed by iGEM, we will use the GFPgenerator (E0240) in association with its RBS (E0032), to caracterise the '''expression of the gene behind a given promoters'''. However, the RBS before the genes which codes for the ''transcriptions factors'' we want to induce, are the natural RBS (specific respectively to ''tetR'', ''flhDC'', ''fliA'', etc... ). Therefore, we must pay attention on what we are measuring.<br />
<br />
The idea is that, by considering that the ''traduction rate'' depends near '''linearly of the Ribosome Binding Site''' (as we guess ; it gives the affinity between the mRNA and the ribosome !), we introduce a constant factor between the ''expected'' value of a protein and its ''real'' value.<br />
<br />
Moreover, we observe that at the steady state, the degradation rate ''&gamma;'' has got a linear effect on the concentration of the protein at equilibrium, too.<br />
<br />
For instance, if we consider a protein ''prot'' coded by its corresponding ''gene'', put behind one of our "caracterised promoters", with its natural RBS, we will have between our construction (''expected'' value, given by GFP) and the ''real'' expression of ''prot'' the following relation<br />
<br />
[[Image:protcoef.jpg|center]] where [[Image:coef.jpg|center]]<br />
<br />
Because we want to stay as near as possible to the "natural construction of the flagella" (see [[Team:Paris/Project|our project]]), we will keep the natural RBS of every genes we want to put in our final construction (particularly ''flhDC'' and ''fliA'').<br />
<br />
Because of the undetermined strength of the RBS and degradation rate of the different proteins, the parameters we will have acces to are biased. When a parameter is noted K<sub>ref</sub> and is referenced later in K<sub>ref</sub>/{coef}, <br />
that means that we will calculate the latter, and that this value will be the one instead of the former in the [[Team:Paris/Modeling/FIFO#Resulting_Equations|equations]] we will introduce in the system of ODE.<br />
<br />
Also we must remember that the we are not necessarly dealing with real quantities, but with ''abstract quantities that are coherents together, in our construction''. Finally, in our research of caraterising the expression of the genes in a so simplified way, those methods allow us to skip evaluations of the relative strength of the RBS, and of the degradation rates of our proteins.<br />
<br />
To conclude, we nevertheless must notice that the effect of the coefficient ''&gamma;'' in our model has got an other effect : it changes the dynamics of the system, in the time in which the ''steady states'' are reached. As our system intend to produce ''oscillations'', the ''transcient states'' are important, and here is the biggest weakness of this model.<br />
<br />
==How to control the concentration of the transcription factor ?==<br />
===Using {aTc, TetR, pTet}===<br />
Now, we must use as a variable of reference an element that could be introduced in the bacteria, well-controlled, and from which all the concentrations of our transcription factor will depend. We propose a construction in which our transcription factor is put after the promoter pTet, which is under the repression of TetR. Since aTc is a small diffusive molecule that binds to TetR and inhibits this way the repression of pTet, we can use it as an "inducer". To do so, we must place in the bacterium the gene ''tetR'' after a constitutive promoter (like J23101).<br />
<br />
We have<br />
<br />
[[Image:ExprTetR.jpg|center]]<br />
<br />
So, according to the calculus on [[Team:Paris/Modeling/Programs#Induction_for_the_caracterisations|the diffusion and complexation]] we will get at the steady-state :<br />
<br />
<br />
[[Image:ExprTetREq.jpg|center]]<br />
<br />
<br />
In the last equation, we will have 'access' (see [[Team:Paris/Modeling/estimation#First_hypothesis|hypothesis '''(5)''']]) to ''expr(pTet)'' and possibly to ''&gamma;'', and we are looking for β<sub>tet</sub>, K<sub>tet</sub> and n<sub>tet</sub>, thanks to our program (see [[Team:Paris/Modeling/estimation#Getting_a_.22complexation_function.22_from_convenient_datas|getting Hill function with convenient datas]]).<br />
We control [aTc]<sub>i</sub> and we will caracterise ''expr(J23101)'' (see [[Team:Paris/Modeling/estimation#What_are_we_looking_for_.3F|the functions we are looking for]]). Then, to solve the equation, we need K<sub>aTc</sub> and n<sub>aTc</sub>.<br />
Therefore, we will first brievly look into the literature, and then complexify the [[Team:Paris/Modeling/Programs#Finding_Parameters|'findparam' function]], in order to seek for these two more parameters. This new program allows us to get [[Team:Paris/Modeling/Programs#Induction_by_a_small_molecule|Prot = fun(mol_dif,rec)]], i.e. a protein expression in function of a small diffusive molecule and of its receptor, acting on a specific promoter.<br />
<br />
===Using {AraC, Arabinose, pBad}===<br />
<br />
An other well-known promoter called pBad, is induced by the complex AraC><arab, where AraC appears to be a protein ''constitutively produced'' by an operon attached to pBad, and arabinose is a sugar, that we can add at will in the medium, and diffuses through cells.<br />
We have :<br />
<br />
[[Image:exprpBad.jpg|center]]<br />
<br />
we find at steady state (with obvious notations, as in the last paragraph)<br />
<br />
[[Image:PbadArab.jpg|center]]<br />
<br />
However, we do not need any estimation of ''const.expr(pBad)'', because we do not have to get the "real" K<sub>bad</sub>, since we will only use this promoter in ''caracterisation systems''. Then, we will treat the other parameters exactly as for the ''pTet system''.<br />
<br />
==What are we looking for ?==<br />
The different functions we would like to determine are the followings. They are linked to a basic description of the "theoretical protocol" that will allow us to get the expected datas. We decided, as far as possible, to keep the original genome of the bacteria, so that the strength that we are measuring is ''' the strength for an "additional" promoter in the cell''', keeping those which already exist : this makes sense for our construction, and probably for most of the constructions of synthetic biology wih ''E.coli''.<br />
<br />
<br />
*[[Team:Paris/Modeling/f0|[expr.(''J23101'')] = &#131;0]]<br />
<br />
*[[Team:Paris/Modeling/f1|[expr.(pTet)] = &#131;1([TetR],[aTc])]]<br />
<br />
*[[Team:Paris/Modeling/f2|[expr.(pBad)] = &#131;2([arabinose])]]<br />
<br />
<br><br />
<br />
*[[Team:Paris/Modeling/f3|[expr.(pFlhDC)] = &#131;3([OmpR*],[FliA])]]<br />
<br />
*[[Team:Paris/Modeling/f3bis|[expr.(pFlhDC)] = &#131;3bis([EnvZ],[FliA])]] <br />
<br />
*[[Team:Paris/Modeling/f4|[expr.(pFlhDC)] = &#131;4([FlhDC],[FliA])]]<br />
<br />
*[[Team:Paris/Modeling/f5|[expr.(pFliL)] = &#131;5([FlhDC],[FliA])]]<br />
<br />
*[[Team:Paris/Modeling/f6|[expr.(pFlgA)] = &#131;6([FlhDC],[FliA])]]<br />
<br />
*[[Team:Paris/Modeling/f7|[expr.(pFlgB)] = &#131;7([FlhDC],[FliA])]]<br />
<br />
*[[Team:Paris/Modeling/f8|[expr.(pFlhB)] = &#131;8([FlhDC],[FliA])]]<br />
<br />
*[[Team:Paris/Modeling/f9|[expr.(pLas)] = &#131;9([LasR><HSL])]]<br />
<br />
= Noise estimation =<br />
Firstly, we could compute the standard-deviation for each set of points at a given inducer concentration, and to normalize it according to the mean value of the set itself.<br />
<br />
The next stage is to get the mean ('''&sigma;''') of those normalized s-d values at every inducer concentration.<br />
<br />
Since the '''β''' parameter (''production rate'') has a linear influence on a production function, it is possible to translate this error directly on the parameter; for example: '''β<sub>stochastic</sub> ~ LG(&beta;,&sigma;)''', where LG denotes a Laplace-Gauss law of probability.<br />
<br />
For each cell in the model, we could use such noised values for Vmax parameter, in order to reproduce randomness estimated in the wet lab.</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Modeling/ProgramsTeam:Paris/Modeling/Programs2008-10-30T07:06:54Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
<br />
==Complexations Caracterisation==<br />
<br />
The first hypothesis is that a complexation reaction is fully determined by the following :<br />
<br />
<center> [[Image:reacthill.jpg]] </center><br />
<br />
and that the rates k<sub>+</sub> et k<sub>-</sub> stay constant under all conditions. Then, the second hypothesis is that these equations are (kinetically speaking) ''elementary'' :<br />
<br />
<center> [[Image:kinhill.jpg]] </center><br />
and at steady-state : <center> [[Image:sshill.jpg]] </center><br />
<br />
Then, since we guess that the only datas we will have are the quantities of ''A'' and ''B'' introduced (''A<sub>i</sub>'' and ''B<sub>i</sub>''), the only equations we will deal with is the following, entirely determining the concentration ''C<sub>eq</sub>'' at steady-state (at least if we take the smallest real root of the equation, it is useless to demonstrate the unicity, or even the existence, of such a solution) :<br />
<br />
<center> [[Image:Eqnss.jpg]] </center><br />
<br />
==Equilibrium of a Complex==<br />
<br />
Know, if we imagine a given amount of ''A<sub>i</sub>'' and ''B<sub>i</sub>'', that are calculated as their equilibrium without taking acount of their complexation (but, for instance, of other interactions, productions and disappearance), and that the produced complex C disappears along time with a ''degradation rate &gamma;'', we get :<br />
<br />
[[Image:GammaC.jpg|center]]<br />
<br />
so that the equilibrium gives :<br />
<br />
[[Image:EqGammaC.jpg|center]]<br />
<br />
with<br />
<br />
[[Image:Keff.jpg|center]]<br />
<br />
==Hill Functions==<br />
<br />
The previous system of complexation applied in particular to the association of the Promoters (''P'') and its Transcription Factor (''TF'').<br />
<br />
Because the promoters on a "low copy plasmid" exists in the cell in ten exemplaries, in contrary to a protein, which, as long as it is produced (even weakly) exists in thousands of exemplaries, we assume can the quantities of ''TF'' and ''P'' are different by several orders of magnitude. Then, with the previous notations, if ''A'', ''B'' and ''C'' stands respectively for ''TF'', ''P'' and the complex ''TF><P'', we will get<br />
<br />
[[Image:approxhill.jpg|center]]<br />
<br />
that we can easily solve with :<br />
<br />
[[Image:reshill.jpg|center]]<br />
<br />
Depending to the order ''' ''n'' ''' (also called ''cooperativity'', because it correponds to the possibilities of the transcription factor to binds in a group on the promoter), this function is a ''sigmoïd'', known as the ''Hill function''. The parameter ''' ''K'' ''', called ''activation constant'', is often replaced in the previous expression by the following notation<br />
<br />
[[Image:changeK.jpg|center]]<br />
<br />
It simplifies the manipulations of the expression ; we can notice that ''' ''K'' ''' represents now the amount of ''TF'' needed to bind half of the total ''P'' in the cell.<br />
<br />
[[Image:hilldef.jpg|center]]<br />
<br />
==Induction by a small molecule==<br />
<br />
===Introduction===<br />
In certain steps of our [[Team:Paris/Project|system]], and in our [[Team:Paris/Modeling/estimation|caracterisation plan]], we use the diffusion of a small molecule (''SM''), that binds to a transcription factor.<br />
<br />
We make the hypothesis of a '''simple, passive diffusion''', that leads at the steady-state at equal amount of the small molecule, inside and outside the cells. The resulting equations are the following coupling :<br />
<br />
[[Image:CouplDiff.jpg|center]]<br />
<br />
where<br />
<br />
* ''permeability'' is the membrane permeability multiplied by the average external surface of a cell (in min<sup>-1</sup>)<br />
* ''N'' is the average cell population<br />
* ''V<sub>int</sub>'' is the average volume of a cell (in L) ; ''V<sub>ext</sub>'' is the volume of the culture medium outer the cells (in L)<br />
<br />
===The situation===<br />
<br />
The involvement of the previous process is, in our systems,under these conditions :<br />
<br />
* ''Prot'' is a protein, produce by a ''promoter''<br />
* ''expr_prom'' is the given expression of this ''promoter'' (production rate of ''Prot'')<br />
* ''&gamma;'' is the degradation/dilution rate of the proteins and complexes in the cell (especially due to cell division, we made so far the hypothesis that it is the same for all the proteins)<br />
* ''SM<sub>int</sub>'' binds to ''Prot'', with a cooperativity ''n'' and a dissociation constant ''K<sub>d</sub>'', to form the complex ''Compl''<br />
* The cell culture is in a chemostat : the cell population is N; The renewal rate (flow of the medium, divided by the volume of the chemostat, in min<sup>-1</sup>) is ''rnw''; The Volume of a cell is ''V<sub>int</sub>''; and of the outer medium is ''V<sub>ext</sub>'' ( = ''Volume(chemostat)'' - ''N'' * ''V<sub>int</sub>'')<br />
<br />
===Induction for the caracterisations===<br />
see [[Team:Paris/Modeling/estimation#How_to_control_the_concentration_of_the_transcription_factor_.3F| details on the induction]]<br />
<br />
In these experimental conditions, we assume that the external concentration of SM remains constant. So, at steady-state, <br />
<center>[''SM'']<sub>ext</sub> = [''SM'']<sub>int</sub></center><br />
<br />
Then, considering the previous complexations equations, and by the equilibrium of ''Prot'' ( ''expr(prom)'' denotes protein production rate ), we have at steady states<br />
<br />
[[Image:ProtSt.jpg|center]]<br />
<br />
and by the definition of the dissociation constant<br />
<br />
[[Image:Kd.jpg|center]]<br />
<br />
that leads to the following expression of the complexes and proteins :<br />
<br />
[[Image:ComplProt.jpg|center]]<br />
<br />
These equations will be used to estimate ''K<sub>d</sub>'' and ''n'', and then to estimate the relative amount of bound or free protein in the cell, for a given amount of ''SM''.<br />
<br />
<br />
<br />
==Finding Parameters==<br />
<br />
|}<br style="clear:both" /></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Modeling/hill_approachTeam:Paris/Modeling/hill approach2008-10-30T07:06:34Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
= Model of the ''APE modelisation'' =<br />
==What kind of Mathematical Simulation ?==<br />
<br />
One of the strength of the synthetic biology is that precise knowledge and caracterisation of certains interactions allow very good predictions and simulations. So, our second model intends to get the best precision in the modelisation, consistent with the simpliest (but still logical) hypothesis possible.<br />
<br />
To determine interactions like Michaëlis-Menten's or Hill's, we start from the basical chemicals equations and try to caracterise their consequences on the behaviour of the system with few parameters. For instance, each ''complexation reactions'' will be caracterised at their steady-state, for all sets of initial concentrations (see [[Team:Paris/Modeling/Programs|complexations]]).<br />
<br />
We decided to use mostly Ordinary Differential Equation approach, at least for the study of the Oscillations and of the FIFO. For the Synchronisation module, we will probably use Probabilistic Differential Equations, in order to introduce the differences between the cells.<br />
<br />
==Bio-Chemical General Assumptions==<br />
<br />
We know that the following equations do not describe properly what ''really'' happens in the cells. For exemple, we know that the transcription factor flhD-flhC is actually an ''hexamere'' FlhD<sub>4</sub>C<sub>2</sub>. But, as we will surely not get access to the ''dissociation constant'' of the ''hexamerisation'', we just treat it as an ''abstract'' inducer protein "FlhDC", with an order (''n'') in its ''complexation caracterisation'' probably around 2*4 = 8 (but perhaps completly different ! ; the estimation of the error by the [[Team:Paris/Modeling/Programs#Finding_Parameters|'findparam']] program will tell us if we are right to do so).<br />
<br />
For the moment, at each part of our modelisation, we reduce the expression of a gene at its '''transcription'''. The '''translation''' process is not taken into acount (see however [[Team:Paris/Modeling/estimation#RBS_Issue|considerations on RBS]]).<br />
Besause of that, our caracterisations doesn't allow us to know the ''real concentrations'' of the proteins we produce, but only their "real times effects" on the promoters they influence.<br />
<br />
Every complexations we deal with are the supposed to reach ''immediatly'' their '''steady-states'''. So, the only phenomenon we are observing along time is the protein production ; that's why this model can't really simulate the possible delay a complexation could introduce, and which could be important for the stiffness of our oscillations...<br />
<br />
As we need to keep our cells in the exponential phase of growth (and since we can't use the already mentioned system of Ron Weiss, see [[Team:Paris/Project|description of the project]]), our system works in a '''chemostat'''. We will also be able to estimate the Cell Density, and we will have to take into acount the ''renewal phenomenon''. Under this conditions, we assume the following constants to be true :<br />
<br />
* Cell Division : every 35 minutes<br />
&rarr; Dilution Rate : 0.0198 min<sup>-1</sup><br />
* Cell Density : cell.L<sup>-1</sup><br />
&rarr; Average Intracellular Volume : L <br><br />
&rarr; Average Extracellular Volume (in the chemostat) : L<br />
* Renewal Rate : L.min<sup>-1</sup><br />
(&rarr; Dilution Rate : min<sup>-1</sup>)<br />
<br />
To see more details about the values of the involved constants, see [[Team:Paris/Modeling/Bibliography|the bibliography]] and the [[Team:Paris/Modeling/estimation|estimation section]].<br />
<br />
==Incrementally detailed Parts of our Project==<br />
<br />
* [[Team:Paris/Modeling/Oscillations|Oscillations]]<br />
<br />
* [[Team:Paris/Modeling/FIFO|FIFO]]<br />
<br />
* [[Team:Paris/Modeling/Synchronisation|Synchronisation]]<br />
<br />
<br style="clear:both" /></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Modeling/BOB/AkaikeTeam:Paris/Modeling/BOB/Akaike2008-10-30T07:06:20Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
<br><br />
<center><html><div style="color:#275D96; font-size:2em;">Depending on the experimental constraints, how can we be helped by <br><br>mathematical criterions ?</div></html></center><br />
<br><br />
* When building a model, it is of the utmost importance to present a justification of the choice made along the transposition process from biological reality to mathematical representation. The aim of this section is to introduce a mathematical justification of our choices in the [[Team:Paris/Modeling/BOB|BOB approach]] since it seems remote from biological reality compared to our [[Team:Paris/Modeling/hill_approach|APE approach]]. The criterion presented below are to help choosing the most reluctant model '''given the experimental constraints'''.<br />
<br><br />
= Short introduction to the criteria =<br />
* Using linear equations in a biological system might seem awkards. However, we wanted to check the relevance of this approach. We have been looking for a criterium that would penalize a system that had many parameters, but that would also penalize a system which quadratic error would be too important while fitting experimental values. The goal here is to decide whether, assuming that the experimental data looks like a model based on Hill functions, the linear part of the BOB model is obsolete or not.<br />
* Akaike and Schwarz criteria taken from the information theory met our demands quite well :<br />
Akaike criterion : [[Image:AIC.jpg|center]]<br />
Hurvich and Tsai criterion : [[Image:AICc.jpg|center]]<br />
Schwarz criterion : [[Image:BIC.jpg|center]]<br />
where n denotes the number of experimental values, k the number of parameters and RSS the residual sum of squares.<br />
The best fitting model is the one for which those criteria are minimized.<br />
* It is remarquable that Akaike criterion and Hurvich and Tsai criterion are alike. AICc is therefore used for small sample size, but converges to AIC as n gets large. Since we will work with 20 points for each experiment, it seemed relevant to present both models. In addition, Schwarz criterion is meant to be more penalizing.<br />
<br />
= Experiment =<br />
* As an experiment, we wished to compare the two models presented below : <br />
'''System#1''' : using the linear equations from our BOB approach :<br><br />
[[Image:syste_akaike_1.jpg|center]]<br><br />
'''System#2''' : using classical Hill functions :<br><br />
[[Image:syste_akaike_2_bis.jpg|center]]<br><br />
* We made a set of data out of a noised Hill function. In fact, our data set was made by using the same equations as System#2, but we introduced a normal noise for each point. Thus, System#1 is penalized because its RSS will be greater than that of System#2. Nevertheless, System#2 will be more penalized by its number of parameters.<br />
* With Matlab, we run a fitting simulation for each system, and we obtained the RSS. We then evaluated the different criteria for both models. We chose to act as if the length of our experimental data set was 20 (which is what we would have gotten in reality). The results are presented below.<br />
<center><br />
{|<br />
|- style="background: #649CD7;"<br />
! colspan="3" style="background: #649CD7;" | Comparison of the systems for n=20<br />
|- style="background: #649CD7; text-align: center;"<br />
| Criteria<br />
| System#1<br />
| System#2<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | AIC<br />
| 26.7654<br />
| 32.0035<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;"| AICc<br />
| 38.7654<br />
| 168.0035<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;"| BIC<br />
| 22.9435<br />
| 24.3596<br />
<br />
|}<br />
<br><br />
<br><br />
{|<br />
|- style="background: #649CD7;"<br />
! colspan="3" style="background: #649CD7;" | Comparison of the systems for n=100<br />
|- style="background: #649CD7; text-align: center;"<br />
| Criteria<br />
| System#1<br />
| System#2<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;" | AIC<br />
| 169.5495<br />
| 32.1150<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;"| AICc<br />
| 171.1147<br />
| 38.5912<br />
|- style="background: #dddddd;" <br />
| style="background: #D4E2EF;"| BIC<br />
| 172.0100<br />
| 37.0360<br />
<br />
|}<br />
<br />
</center><br />
<br />
* Finally, we wanted to check that those numerical simulation did not hide anything. Then, we run the simulation plots obtained for the parameters that best fitted. Here are the results :<br />
<br />
[[Image:plotRef.jpg|center|Reference (noised Hill)]]<br />
[[Image:plotAkaikeBob.jpg|400px|left|thumb|Bob Model]]<br />
[[Image:plotAkaikeHill.jpg|400px|right|thumb|Hill Model]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>Then, we see that the Bob model does not fit that well, but under certain conditions it is not so bad. Indeed, if the data set is short, Bob model can be used as a reluctant model, that helps understanding the dynamics.<br />
<br />
* Consequently, what have we proved? These results show that:<br />
** Firstly, we may see that the AICc does converge to AIC for greater values of n.<br />
** Then, we may see that, as predicted, System#2 is not penalized anymore for greater values of n, although System#1 is. Consequently, we may adapt our system knowing the length of the data set we are going to obtain experimentally.<br />
** Furthermore, since the use of more parameters is quite penalizing for a small set of data, and since the criteria are minimized for System#1, the first subsystem of our BOB model is not irrelevant. <br />
** However, since for a larger set of data System#2 minimizes the criteria, these criteria cannot decide whether a model is "better" than another one, since those criteria are arbitrary. Yet, they may help us find a better compromise between simplification and accuracy.<br />
** One must be careful when building a model, since chosing the number of parameters and deciding how deep one wishes to go into detail, influences the goal and the results of a model. It is therefore important to understand that a model has to be conceived to achieve a precise aim.<br />
** Then, it is always useful to use different models, knowing that each model meets a certain demand. Here, our full model ([[Team:Paris/Modeling/hill_approach|APE]]) will be used to present a highly detailed overview of the processes that take place in the system. In the mean time, the [[team:Paris/Modeling/BOB|BOB]] approach is a reasonable mean to explore the system quickly and understand its ins and outs.<br />
<br />
= A fundamental tool? = <br />
Why can we introduce this seemingly awkard criteria as being a fundamental tool? This precise criteria enables the mathematician to adapt its model. In fact, in that respect, conducting this analysis over his model gives tangible arguments to the mathematician to use such and such model. Indeed, for example in our precise case, if we have about 20 experimental points to fit, BOB approach is sufficient. However, if we get 50 points, BOB approach would be inadequate compared to APE. We believe that this kind of criteria is an essential tool, that might help the model maker to comprehend and control the assumptions he made while creating his model.<br />
<br />
----<br />
* We mostly used the definition of the criteria given in :<br />
[http://www.liebertonline.com/doi/pdf/10.1089/rej.2006.9.324 K. Kikkawa.''Statistical issue of regression analysis on development of an age predictive equation''. Rejuvenation research, Volume 9, n°2, 2006.]<br />
* [[Team:Paris/Modeling/BOB/Simulations/Codes#Model_justification|Here]] can be found the programs used in this section.</div>Romain.rousseauhttp://2008.igem.org/Template:Paris/MenuTemplate:Paris/Menu2008-10-30T07:03:47Z<p>Romain.rousseau: </p>
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|style="background:#fff"|</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-30T07:00:12Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Presentation of the project}}<br />
{{Paris/Section_contents_project}}<br />
= Outlines =<br />
[[Image:Specmontre.jpg|right|250 px]]<br />
<br />
Our BacteriO'Clock project aims at constructing a '''First In First Out (FIFO) periodical system synchronized at population scale'''. To this end we implement a biological FIFO circuit in a negative feedback loop and a cell-cell communication channel. Such a setup is designed to trigger a sequence of several genes successive activations repeated periodically. Thus, it can be considered as a biological clock. <br />
<br />
* '''FIFO behavior''' : Our setup involves three genes which will get activated and deactivated successively following a “FIFO : First In, First Out” order. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).We base this part of our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella. What is most interesting in a FIFO is the study of the ON and OFF steps. An easier way to observe the FIFO would be to have automatic oscillations.<br />
<br />
* '''Oscillatory behavior''' : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer. The period of the oscillations is even more interesting if it allows the sequential switching on and off of several genes.<br />
<br />
* '''Synchronization''' : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
[[Image:Queue2.jpg|left|thumb|300px|A queue in front of the Post Office traditionally works as a FIFO : the person arrived first will be the first served, and will ''probably'' leave the Post Office first.]]<br />
<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">''If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer. You would accumulate unprocessed intermediates !''</span><br />
<br />
<br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO.<br />
<br><br><br />
<br />
For more details, see here :<br />
*[[Team:Paris/Analysis/Design1|Explanations for the FIFO]]<br />
<br />
== Oscillations ==<br />
[[Image:Oscill.png|right|thumb|250px]]<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead in theory to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology.<br />
<br />
For more details, see here :<br />
*[[Team:Paris/Analysis/Design2|Details about the oscillations]]<br />
<br />
== Synchronization ==<br />
[[Image:synchro.jpg|left|thumb|250px]]<br />
<br />
In order to make visualization easier and to permit the development of future applications, our system must reach a higher scale. As a consequence, the synchronization of the oscillation at a population level is a key issue in our system. The classical way to do so, is to introduce a ''checkpoint'' at the end of each cycle, as in the eukariotic cell cycle.<br />
<br />
Several teams around the world worked on the synchronization of oscillations. The phenomenon of ''quorum-sensing'', is a natural way to synchronize the expression of gene network in bacteria. We will embezzle a natural ''quorum sensing'' system to do so.<br />
<br />
For more details, see here :<br />
*[[Team:Paris/Analysis/Design3|Details about the synchronization]]<br />
<br />
{{Paris/Navig|Team:Paris/Project}}</div>Romain.rousseauhttp://2008.igem.org/Template:Paris/Section_contents_characterizationTemplate:Paris/Section contents characterization2008-10-30T06:58:21Z<p>Romain.rousseau: </p>
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<ul><br />
<li><a href="https://2008.igem.org/Team:Paris/Modeling/Workflow_Example">Characterization Approach : An Example</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Modeling/Molecular_Reactions">Basic Molecular Reactions</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Modeling/FromMolReactToNLOde">Bio-Mathematical Modeling</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Modeling/Protocol_Of_Characterisation">Protocol of Characterization</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Modeling/Implementation">Implementation</a></li><br />
</ul><br />
</div><br />
</html></div>Romain.rousseauhttp://2008.igem.org/Template:Paris/Section_contents_analysisTemplate:Paris/Section contents analysis2008-10-30T06:57:28Z<p>Romain.rousseau: </p>
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<li><a href="https://2008.igem.org/Team:Paris/Analysis/Construction">Model Construction</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Math%2BSim#FIFO">Mathematical Analysis and Simulations</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Construction2">System Improvements Description</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Model2_Analysis">System Improvements Analysis</a></li><br />
</ul><br />
</div><br />
</html></div>Romain.rousseauhttp://2008.igem.org/Template:Paris/Section_contents_projectTemplate:Paris/Section contents project2008-10-30T06:56:23Z<p>Romain.rousseau: </p>
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<span>Other pages:</span><br />
<ul><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design1">The FIFO Behaviour</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design2">Oscillations</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design3">The synchronization Module</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design3#Global_genetic_network">Global Genetic Network</a></li><br />
</ul><br />
</div><br />
</html></div>Romain.rousseauhttp://2008.igem.org/Template:Paris/Section_contents_projectTemplate:Paris/Section contents project2008-10-30T06:55:34Z<p>Romain.rousseau: </p>
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<span>Other pages:</span><br />
<ul><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design1">The FIFO Behaviour</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design2">Oscillations</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design3">The synchronization Module</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design3#Final_genetic_network">Global Genetic Network</a></li><br />
</ul><br />
</div><br />
</html></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Network_Design2Team:Paris/Network Design22008-10-30T06:55:17Z<p>Romain.rousseau: Team:Paris/Network Design2 moved to Team:Paris/Analysis/Design2</p>
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<div>#REDIRECT [[Team:Paris/Analysis/Design2]]</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Analysis/Design2Team:Paris/Analysis/Design22008-10-30T06:55:17Z<p>Romain.rousseau: Team:Paris/Network Design2 moved to Team:Paris/Analysis/Design2</p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Network Design - Part 2}}<br />
{{Paris/Section_contents_project}}<br />
<br />
=Creating an oscillating system=<br />
== Already existing genetic oscillators and their limits ==<br />
Designing a simple genetic network that presents an oscillatory behavior is one of the first challenge synthetic biology overcame. More or less successfully. We can count more than ten synthetic genetic oscillators that have varied period and mechanisms. [http://www.ncbi.nlm.nih.gov/pubmed/16604190?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Raúl GUANTES and Juan F. POYATOS (2006)] studied the most simple oscillators composed of two elements while [http://www.ncbi.nlm.nih.gov/pubmed/10659856?ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Michael B. ELOWITZ and Stanislas LEIBLER (2000)] designed the more complex "''repressilator''" (''Table 1''), to quote only the best known.<br />
<br />
[[Image:Oscillators.jpg|thumb|760px|center|Tab 1. Two examples of genetic oscillators. A : a simple oscillator composed of two elements. B : the repressilator. (Legend : Green arrow : Activation. Red blunt arrow : Inhibition)]]<br />
<br />
Both oscillators work : we can observe oscillations but only a limited number of cycles. Actually, they always reach a steady-state because the degradation/dilution rate is often too low : at the end of each cycle, the conditions are not exactly the initial conditions. Experimentally, '''the longer is the period the more cycles we can observe'''.<br />
<br />
== Design of our genetic oscillator : The Feed Forward Loop ==<br />
We want to design a simple oscillator that oscillates during as many cycles as possible. We propose a system based on an oscillator composed of two elements (''Network 1'') on which we added a delay at the end of each cycle.<br />
[[Image:Reseauoscillsimple.png|thumb|center|Network 1. Simple oscillator composed of two elements]]<br />
Uri ALON described genetic network motifs that generate a delay. Those motifs are the type 1 coherent Feed Forward Loop (C1-FFL). <br />
<br />
{{Paris/Toggle|More on Feed Forward Loop|Paris/FFL}}<br />
<br />
We will use one of those network to increase the run of each period and permit more oscillations (''Network 2'').<br />
[[Image:Reseauoscill.png|center|thumb|250 px|Network 2. Our oscillator : a C1-FFL increase the length of each period]]<br />
<br />
== Implementation of the core system ==<br />
[http://www.ncbi.nlm.nih.gov/pubmed/16729041?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Shiraz Kalir ''et al.'' (2004)] studied the complex network of gene that lead to the synthesis of ''E. coli'' flagella. <br />
A C1-FFL is present in this network. <br />
{{Paris/Toggle|Click here to know more about ''E. coli'' flagella.|Team:Paris/Flagella}}<br />
*X is flhDC, the master regulator of the synthesis of the flagella. It is associated to its natural promoter. <br />
*Y is fliA, another transcription factor that regulates the expression of a large amount of flagellar genes. fliA is also associated with its natural promoter. <br />
*For Z, we need a protein that inhibits the expression of flhDC. We considered two possibilities. First of all, we planned to use tetR, and the appropriate promoter before flhDC. But, once again, in a concern to use as many "natural" BioBricks, we decided to use proteins that naturally inhibits the expression of flhDC, such as envZ and ompR. The mechanism of inhibition is quite well-known. envZ phosphorylates OmpR which becomes active. OmpR-P strongly inhibits the expression of flhDC. [http://www.ncbi.nlm.nih.gov/pubmed/18193944?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Antoine Giraud ''et al.''(2008)] discribed a specific mutant, '''envZ* that phosphorylates very efficiently ompR'''. We chose envZ* for Z because '''it introduces another delay''', as envZ* works through over-phophorylation of ompR, which in turns represses flhDC. For Z promoter, we chose one of the promoter controlling the expression of one of the flagellar gene that are regulated by both FliA and FlhDC. We chose the promoter of FlhB because it is the gene that is lastly activated. As a consequence, it increases the length of each cycle. Of course, to make the oscillations observable, we decided to put EnvZ and GFP under the control of the same promoter.<br />
[[Image:Reseauoscillfinal.png|center|thumb|250 px|Network 3. Final Design of the core system.]]<br />
<br />
== Limits of our network ==<br />
Intuitively, it seems that there is a range of parameters that permits oscillations. However, [[Team:Paris/Network_analysis_and_design/Core_system#Oscillations|an analysis]] of the core system highlighted the fact that it could hardly have an oscillating dynamics ([[Team:Paris/Network_analysis_and_design/Core_system#Oscillations|see figure: Simulation of the core system]]).<br />
<br />
Among the [[Team:Paris/Network_analysis_and_design/Core_system#System_Improvements|alternatives]] we studied, the system that could most probably oscillate is a [[Team:Paris/Network_analysis_and_design/System_improvements/Analysis#HSL mediated simple oscillator|HSL mediated simple oscillator]] that uses the quorum sensing to produce both a delay and the synchronization at the population level.<br />
<br />
<br />
{{Paris/Navig|Team:Paris/Project}}</div>Romain.rousseauhttp://2008.igem.org/Template:Paris/Section_contents_projectTemplate:Paris/Section contents project2008-10-30T06:54:53Z<p>Romain.rousseau: </p>
<hr />
<div><html><br />
<div id="links"><br />
<span>Other pages:</span><br />
<ul><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design1">The FIFO Behaviour</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Network_Design2">Oscillations</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design3">The synchronization Module</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design3#Final_genetic_network">Global Genetic Network</a></li><br />
</ul><br />
</div><br />
</html></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-30T06:51:57Z<p>Romain.rousseau: </p>
<hr />
<div>{{Paris/Menu}}<br />
{{Paris/Header|Presentation of the project}}<br />
{{Paris/Section_contents_project}}<br />
= Outlines =<br />
[[Image:Specmontre.jpg|right|250 px]]<br />
<br />
Our BacteriO'Clock project aims at constructing a '''First In First Out (FIFO) periodical system synchronized at population scale'''. To this end we implement a biological FIFO circuit in a negative feedback loop and a cell-cell communication channel. Such a setup is designed to trigger a sequence of several genes successive activations repeated periodically. Thus, it can be considered as a biological clock. <br />
<br />
* '''FIFO behavior''' : Our setup involves three genes which will get activated and deactivated successively following a “FIFO : First In, First Out” order. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).We base this part of our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella. What is most interesting in a FIFO is the study of the ON and OFF steps. An easier way to observe the FIFO would be to have automatic oscillations.<br />
<br />
* '''Oscillatory behavior''' : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer. The period of the oscillations is even more interesting if it allows the sequential switching on and off of several genes.<br />
<br />
* '''Synchronization''' : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
[[Image:Queue2.jpg|left|thumb|300px|A queue in front of the Post Office traditionally works as a FIFO : the person arrived first will be the first served, and will ''probably'' leave the Post Office first.]]<br />
<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">''If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer. You would accumulate unprocessed intermediates !''</span><br />
<br />
<br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO.<br />
<br><br><br />
<br />
For more details, see here :<br />
*[[Team:Paris/Network_Design1|Explanations for the FIFO]]<br />
<br />
== Oscillations ==<br />
[[Image:Oscill.png|right|thumb|250px]]<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead in theory to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology.<br />
<br />
For more details, see here :<br />
*[[Team:Paris/Network_Design2|Details about the oscillations]]<br />
<br />
== Synchronization ==<br />
[[Image:synchro.jpg|left|thumb|250px]]<br />
<br />
In order to make visualization easier and to permit the development of future applications, our system must reach a higher scale. As a consequence, the synchronization of the oscillation at a population level is a key issue in our system. The classical way to do so, is to introduce a ''checkpoint'' at the end of each cycle, as in the eukariotic cell cycle.<br />
<br />
Several teams around the world worked on the synchronization of oscillations. The phenomenon of ''quorum-sensing'', is a natural way to synchronize the expression of gene network in bacteria. We will embezzle a natural ''quorum sensing'' system to do so.<br />
<br />
For more details, see here :<br />
*[[Team:Paris/Network_Design3|Details about the synchronization]]<br />
<br />
{{Paris/Navig|Team:Paris/Project}}</div>Romain.rousseauhttp://2008.igem.org/Template:Paris/Section_contents_projectTemplate:Paris/Section contents project2008-10-30T06:51:28Z<p>Romain.rousseau: </p>
<hr />
<div><html><br />
<div id="links"><br />
<span>Other pages:</span><br />
<ul><br />
<li><a href="https://2008.igem.org/Team:Paris/Network_Design1">The FIFO Behaviour</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Network_Design2">Oscillations</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Network_Design3">The synchronization Module</a></li><br />
<li><a href="https://2008.igem.org/Team:Paris/Analysis/Design3#Final_genetic_network">Global Genetic Network</a></li><br />
</ul><br />
</div><br />
</html></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Parts/PlastestTeam:Paris/Parts/Plastest2008-10-30T06:50:07Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
= Plasmid for promoter Amplification & Measurement =<br />
<br />
<br />
[[Image:RBS+.jpg|thumb|RBS +]]<br />
[[Image:RBS-.jpg|thumb|RBS -]]<br />
<br />
Those plasmids are very useful vectors to amplify promoters and to measure their forces using Standard Promoter Units.<br />
The principle is easy to understand.<br />
The plasmid contains the BioBrick restriction sites (EcoRI, XbaI, SpeI and PstI) followed by the standard GFP Tripart (E0240). <br />
Instead of using a big biobrick containing Promoter, RBS, GFP and Terminators, the Biobrick used for the test is only the Promoter. <br />
This plasmid can be used to amplify a promoter because only the promoter is in the biobrick.<br />
<br />
One derivated plasmid do not have the RBS B0032 outside the BioBrick site, it allows to test the influence of the RBS in changing it in the BioBrick. To do so, [[team:Paris/Notebook/Oligo|O142]] does not contains the RBS, the PCR will begin directly at the ATG codon.<br />
<br />
== Oligonucleotides design ==<br />
<br />
Creation of 3 oligonucleotides ([[team:Paris/Notebook/Oligo|O140, O141, O142]]) in order to create two particular plasmids.<br />
[[image:oligo.jpg|frame|center|Design of the oligonucleotides [[team:Paris/Notebook/Oligo|O140 and O141]] ]]<br />
<br />
== Construction of the Plasmid ==<br />
Necessary material<br />
* Oligos O140, O141 and O142<br />
* E0240<br />
* pSB3K3</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/PartsTeam:Paris/Parts2008-10-30T06:48:08Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
<br />
[[Team:Paris/Parts2|Not in the registry]]<br />
=Parts Submitted to the Registery=<br />
==Basic Parts==<br />
===[[Image:Part_icon_regulatory.png]] Regulatory===<br />
<br />
====Promoter of fliL gene from ''E.coli'' flagella====<br />
*[http://partsregistry.org/Part:BBa_K136000 BBa_K136000]<br />
*Sequencing : NO<br />
*References :<br />
[1] Kalir S, McClure J, Pabbaraju K, Southward C, Ronen M, Leibler S, Surette MG, Alon U. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science. 2001 Jun 15;292(5524):2080-3.<br />
<br />
[2] Shiraz Kalir, Uri Alon. Using quantitative blueprint to reprogram the dynamics of the flagella network. Cell, June 11, 2004, Vol.117, 713-720<br />
<br />
[3] Kalir S, Mangan S, Alon U. A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Mol Syst Biol. 2005;1:2005.0006. Epub 2005 Mar 29.<br />
<br />
====Promoter of flgA gene from ''E. coli'' flagella====<br />
*[http://partsregistry.org/Part:BBa_K136001 BBa_K136001]<br />
*Sequencing : OK<br />
*References :<br />
[1] Kalir S, McClure J, Pabbaraju K, Southward C, Ronen M, Leibler S, Surette MG, Alon U. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science. 2001 Jun 15;292(5524):2080-3.<br />
<br />
[2] Shiraz Kalir, Uri Alon. Using quantitative blueprint to reprogram the dynamics of the flagella network. Cell, June 11, 2004, Vol.117, 713-720<br />
<br />
[3] Kalir S, Mangan S, Alon U. A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Mol Syst Biol. 2005;1:2005.0006. Epub 2005 Mar 29.<br />
<br />
====Promoter of flgB gene from ''E. coli'' flagella====<br />
*[http://partsregistry.org/Part:BBa_K136002 BBa_K136002]<br />
*Sequencing : NO<br />
*References :<br />
[1] Kalir S, McClure J, Pabbaraju K, Southward C, Ronen M, Leibler S, Surette MG, Alon U. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science. 2001 Jun 15;292(5524):2080-3.<br />
<br />
[2] Shiraz Kalir, Uri Alon. Using quantitative blueprint to reprogram the dynamics of the flagella network. Cell, June 11, 2004, Vol.117, 713-720<br />
<br />
[3] Kalir S, Mangan S, Alon U. A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Mol Syst Biol. 2005;1:2005.0006. Epub 2005 Mar 29.<br />
====Promoter of flhB gene from ''E. coli'' flagella====<br />
*[http://partsregistry.org/Part:BBa_K136003 BBa_K136003]<br />
*Sequencing : NO<br />
*References :<br />
[1] Kalir S, McClure J, Pabbaraju K, Southward C, Ronen M, Leibler S, Surette MG, Alon U. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science. 2001 Jun 15;292(5524):2080-3.<br />
<br />
[2] Shiraz Kalir, Uri Alon. Using quantitative blueprint to reprogram the dynamics of the flagella network. Cell, June 11, 2004, Vol.117, 713-720<br />
<br />
[3] Kalir S, Mangan S, Alon U. A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Mol Syst Biol. 2005;1:2005.0006. Epub 2005 Mar 29.<br />
====Promoter of flhDC gene from ''E. coli'' flagella====<br />
*[http://partsregistry.org/Part:BBa_K136004 BBa_K136004]<br />
*Sequencing : NO<br />
*References :<br />
[1] Kalir S, McClure J, Pabbaraju K, Southward C, Ronen M, Leibler S, Surette MG, Alon U. Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science. 2001 Jun 15;292(5524):2080-3.<br />
<br />
[2] Shiraz Kalir, Uri Alon. Using quantitative blueprint to reprogram the dynamics of the flagella network. Cell, June 11, 2004, Vol.117, 713-720<br />
<br />
[3] Kalir S, Mangan S, Alon U. A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Mol Syst Biol. 2005;1:2005.0006. Epub 2005 Mar 29.<br />
<br />
=Parts not Submitted to the Registery=<br />
===[[Image:Icon_coding.png]] Protein coding===<br />
====FlhDC gene from ''E.coli'' flagella with natural RBS====<br />
*[http://partsregistry.org/Part:BBa_K136005 BBa_K136005]<br />
<br />
====Coding sequence for FliA with RBS====<br />
====Coding sequence for FlhDC without RBS====<br />
====Coding sequence for FliA without RBS====<br />
====Coding sequence for EnvZ*====<br />
====Coding sequence for OmpR*====<br />
<br />
==Intermediate Constructions==<br />
==Finished Constructions==</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Project/SynchronisationTeam:Paris/Project/Synchronisation2008-10-30T06:47:56Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
<br />
= First Model =<br />
<br />
[[Image:Synchro1.jpg|500px|center]]<br />
<br />
Strain : double mutant ΔflhDC and ΔflgM <br />
<br />
Designed plasmids<br />
<center><br />
{| border="1" <br />
|align="center"|Generic Name<br />
|align="center"|Construction<br />
|align="center"|Advancement<br />
|-<br />
|align="center"|FlhDC<br />
|align="center"|pTet - FlhDC<br />
|align="center"|☣<br />
|-<br />
|align="center"|AHL enzyme<br />
|align="center"|pFlhB - LasI<br />
|align="center"|☣<br />
|-<br />
|align="center"|AHL Receptor<br />
|align="center"|pConstitutive - LasR<br />
|align="center"|☣<br />
|-<br />
|align="center"|FlhDC Inhibitor<br />
|align="center"|pLas - TetR - GFP<br />
|align="center"|☣<br />
|}<br />
</center><br />
<br />
= Second Model =<br />
<br />
<br />
<br />
[[Image:Synchro2.jpg|500px|center]]<br />
<br />
<br />
Strain : mutant ΔflgM <br />
<br />
Designed plasmids<br />
<center><br />
{| border="1" <br />
|align="center"|Generic Name<br />
|align="center"|Construction<br />
|align="center"|Advancement<br />
|-<br />
|align="center"|Reporter<br />
|align="center"|pFlhB - LasI<br />
|align="center"|☣<br />
|-<br />
|align="center"|AHL Receptor<br />
|align="center"|pConstitutive - LasR<br />
|align="center"|☣<br />
|-<br />
|align="center"|FlhDC Inhibitor<br />
|align="center"|pLas - OmpR - GFP<br />
|align="center"|☣<br />
|}<br />
</center></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Project/FIFOTeam:Paris/Project/FIFO2008-10-30T06:47:43Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
<br />
[[image:FIFObis.jpg|500px|center]]<br />
<br><br><br />
Strain : double mutant ΔflhDC and ΔflgM <br />
if possible tetR :: Tn10<br />
<br />
Designed plasmids<br />
<br><br />
<center><br />
{| border="1" <br />
|align="center"|'''Generic Name'''<br />
|align="center"|'''Construction'''<br />
|align="center"|'''Advancement'''<br />
|-<br />
|align="center"|Master regulator<br />
|align="center"|pTet – FlhDC<br />
|align="center"|☣<br />
|-<br />
|align="center"|Reporter1<br />
|align="center"|pFliL - CFP<br />
|align="center"|☣<br />
|-<br />
|align="center"|Reporter2<br />
|align="center"|pFlgA(B) - YFP<br />
|align="center"|☣<br />
|-<br />
|align="center"|Reporter3<br />
|align="center"|pFlhB - RFP<br />
|align="center"|☣<br />
|}<br />
</center></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/Project/OscillationsTeam:Paris/Project/Oscillations2008-10-30T06:47:31Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
<br />
= First Model =<br />
<br><br />
[[image:Oscillations1.jpg|500px|center]]<br />
<br><br><br />
Strain : double mutant ΔflhDC and ΔflgM (fliA and its promotor is actually on the bacterial chromosome)<br />
<br />
Designed plasmids<br />
<br><br />
<center><br />
{| border="1" <br />
|align="center"|Generic Name<br />
|align="center"|Construction<br />
|align="center"|Advancement<br />
|-<br />
|align="center"|FlhDC<br />
|align="center"|pTet - FlhDC<br />
|align="center"|☣<br />
|-<br />
|align="center"|Reporter<br />
|align="center"|pFlhB - TetR - GFP<br />
|align="center"|☣<br />
|}<br />
<br><br />
</center><br />
<br />
= Second Model =<br />
<br>[[image:Oscillations2.jpg|500px|center]]<br><br><br />
Strain : mutant ΔflgM (fliA and flhDC and their promotors are actually on the bacterial chromosome)<br />
<br />
Designed plasmids<br />
<br><br />
<center><br />
{| border="1" <br />
|align="center"|Generic Name<br />
|align="center"|Construction<br />
|align="center"|Advancement<br />
|-<br />
|align="center"|Reporter<br />
|align="center"|pFlhB - OmpR - GFP<br />
|align="center"|☣<br />
|}<br />
<br><br />
</center></div>Romain.rousseauhttp://2008.igem.org/Team:Paris/ProjectTeam:Paris/Project2008-10-30T06:47:16Z<p>Romain.rousseau: </p>
<hr />
<div>{{:Team:Paris/MenuBackup}}<br />
{{Paris/Header|Presentation of the project}}<br />
{{Paris/Section_contents_project}}<br />
= Outlines =<br />
[[Image:Specmontre.jpg|right|250 px]]<br />
<br />
Our BacteriO'Clock project aims at constructing a '''First In First Out (FIFO) periodical system synchronized at population scale'''. To this end we implement a biological FIFO circuit in a negative feedback loop and a cell-cell communication channel. Such a setup is designed to trigger a sequence of several genes successive activations repeated periodically. Thus, it can be considered as a biological clock. <br />
<br />
* '''FIFO behavior''' : Our setup involves three genes which will get activated and deactivated successively following a “FIFO : First In, First Out” order. This sequence is monitored by a logic structure called Feed-Forward Loop (FFL).We base this part of our project on an already existing structure, partly fulfilling the evoked specifications: the system that leads to the production of ''E. coli'' flagella. What is most interesting in a FIFO is the study of the ON and OFF steps. An easier way to observe the FIFO would be to have automatic oscillations.<br />
<br />
* '''Oscillatory behavior''' : It will consist in providing a periodic output for the duration of the experiment. To do so, we will use a genetic cascade, initiated by a specific inducer which last step will inhibit the previously mentioned inducer. The period of the oscillations is even more interesting if it allows the sequential switching on and off of several genes.<br />
<br />
* '''Synchronization''' : Yet, being able to control this sequential activation within a single cell can be seen as a “first step” in biological clock devising. In order to amplify this phenomenon (to observe it in an easier way or even to find future applications), it has to be extended to a whole population of bacteria. Here comes the synchronization issue: we will use methods based on the “quorum sensing” phenomenon.<br />
<br />
= Motivations =<br />
<br />
== FIFO ==<br />
<br />
[[Image:Queue2.jpg|left|thumb|300px|A queue in front of the Post Office traditionally works as a FIFO : the person arrived first will be the first served, and will ''probably'' leave the Post Office first.]]<br />
<br />
<br />
First In First Out (FIFO) systems are present everywhere from flux management or electronics to genetic networks. A queue in front of the post office works as a FIFO. More generally, it is interesting in any process that requires several steps in a defined order. FIFO behavior indeed prevents from needlessly performing the first steps while the last ones are OFF.<br />
<br />
<span style="font-size:10; color:grey;">''If you want to make French fries you need to produce potatoes before you can cut them and you need to cut them before frying them. But it would be a waste to continue producing potatoes if you've already turned off the fryer. You would accumulate unprocessed intermediates !''</span><br />
<br />
<br />
<br />
The same goes for the bacterium flagella. To be efficient they naturally need to produce the proteins of the base first. But when you stop making flagella, the base proteins are also the first thing you need to stop producing. It has been proposed [http://www.ncbi.nlm.nih.gov/pubmed/15186773?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| (Kalir S., Alon U. 2004)] that the gene network controlling the production of E.coli flagellum behaves as a FIFO. We thus decided to use this regulatory network to implement our FIFO.<br />
<br><br><br />
<br />
For more details, see here :<br />
*[[Team:Paris/Network_Design1|Explanations for the FIFO]]<br />
<br />
== Oscillations ==<br />
[[Image:Oscill.png|right|thumb|250px]]<br />
Genetic oscillators based on the interaction of a small set of molecular components have been shown to be involved in the regulation of the cell cycle, the circadian rhythms, or the response of several signaling pathways. Uncovering the functional properties of such oscillators then becomes important for the understanding of these cellular processes and for the characterization of fundamental properties of more complex clocks.<br />
<br />
Creating an oscillating device has always been at the core of synthetic biology. There are many systems that can lead in theory to this complex behavior with different properties : number of cycles, oscillating period and robustness. Our oscillator has an original structure that has not been tested yet, based on a particular gene network called Feed Forward Loop.<br />
<br />
Engineering and tuning such complex cellular behaviors is a real challenge for synthetic biology.<br />
<br />
For more details, see here :<br />
*[[Team:Paris/Network_Design2|Details about the oscillations]]<br />
<br />
== Synchronization ==<br />
[[Image:synchro.jpg|left|thumb|250px]]<br />
<br />
In order to make visualization easier and to permit the development of future applications, our system must reach a higher scale. As a consequence, the synchronization of the oscillation at a population level is a key issue in our system. The classical way to do so, is to introduce a ''checkpoint'' at the end of each cycle, as in the eukariotic cell cycle.<br />
<br />
Several teams around the world worked on the synchronization of oscillations. The phenomenon of ''quorum-sensing'', is a natural way to synchronize the expression of gene network in bacteria. We will embezzle a natural ''quorum sensing'' system to do so.<br />
<br />
For more details, see here :<br />
*[[Team:Paris/Network_Design3|Details about the synchronization]]<br />
<br />
{{Paris/Navig|Team:Paris/Project}}</div>Romain.rousseauhttp://2008.igem.org/Team:Paris/dumb_testTeam:Paris/dumb test2008-10-30T06:41:32Z<p>Romain.rousseau: Team:Paris/dumb test moved to Talk:Team:Paris over redirect</p>
<hr />
<div>#REDIRECT [[Talk:Team:Paris]]</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:41:32Z<p>Romain.rousseau: Team:Paris/dumb test moved to Talk:Team:Paris over redirect</p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team :) !! - The whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain<br />
<br />
'''This discussion page can be edited at any time so feel free to write what you want.<br />If you are not from our team, welcome here! You can also write any comments about our work.'''</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:41:05Z<p>Romain.rousseau: Talk:Team:Paris moved to Team:Paris/dumb test</p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team :) !! - The whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain<br />
<br />
'''This discussion page can be edited at any time so feel free to write what you want.<br />If you are not from our team, welcome here! You can also write any comments about our work.'''</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:40:30Z<p>Romain.rousseau: </p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team :) !! - The whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain<br />
<br />
'''This discussion page can be edited at any time so feel free to write what you want.<br />If you are not from our team, welcome here! You can also write any comments about our work.'''</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:39:21Z<p>Romain.rousseau: </p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team ;) - The whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain<br />
<br />
'''This discussion page can be edited at any time so feel free to write what you want.<br />If you are not from our team, welcome here! You can also write any comments about our work.'''</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:38:03Z<p>Romain.rousseau: </p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team, the whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain ;)<br />
<br />
'''This discussion page can be edited at any time so feel free to write what you want.<br />If you are not from our team, welcome here! You can also write any comments about our work.'''</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:37:50Z<p>Romain.rousseau: </p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team, the whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain ;)<br />
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This discussion page can be edited at any time so feel free to write what you want.<br />If you are not from our team, welcome here! You can also write any comments about our work</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:37:41Z<p>Romain.rousseau: </p>
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<div>'''Thanks everyone from the iGEM Paris team, the whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain ;)<br />
<br />
This discussion page can be edited at any time so feel free to write what you want.<br />If you are not from our team, welcome here! You can also write any comments about our work</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:37:31Z<p>Romain.rousseau: </p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team, the whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain ;)<br />
<br />
This discussion page can be edited at any time so feel free to write what you want.<br />
If you are not from our team, welcome here! You can also write any comments about our work</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:37:21Z<p>Romain.rousseau: </p>
<hr />
<div>'''Thanks everyone from the iGEM Paris team, the whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain ;)<br />
<br />
This discussion page can be edited at any time so feel free to write what you want. If you are not from our team, welcome here! You can also write any comments about our work</div>Romain.rousseauhttp://2008.igem.org/Talk:Team:ParisTalk:Team:Paris2008-10-30T06:37:06Z<p>Romain.rousseau: New page: '''Thanks everyone from the iGEM Paris team, the whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain ;) (This discussion page can be edited at any time so ...</p>
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<div>'''Thanks everyone from the iGEM Paris team, the whole wiki is frozen since 3 minutes (2008.10.30 @ 07:33 GMT+1/Paris).''' -- Romain ;)<br />
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(This discussion page can be edited at any time so feel free to write what you want. If you are not from our team, welcome here! You can also write any comments about our work)</div>Romain.rousseau