http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&feed=atom&action=history
Team:Slovenia/TheSwitchMutualRepressorSwitch - Revision history
2024-03-29T09:22:40Z
Revision history for this page on the wiki
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http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=289482&oldid=prev
Strazkosann at 20:49, 26 October 2012
2012-10-26T20:49:21Z
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 20:49, 26 October 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchDesignedTALregulators'><span>Designed TAL regulators</span></a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch'><table <ins class="diffchange diffchange-inline">onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchPositiveFeedbackLoopSwitch';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Positive feedback loop switch</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TheSwitchControls'><table <ins class="diffchange diffchange-inline">onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TheSwitchControls';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Controls</span></td><td class="newtable"><img style="margin-right:-81px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </ul></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsEscapeTag'><span>Escape tag</span></a></li> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/SafetyMechanismsTermination'><span>Termination</span></a></li> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href=<del class="diffchange diffchange-inline">'</del>https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation<del class="diffchange diffchange-inline">'</del>><table class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href=<ins class="diffchange diffchange-inline">"</ins>https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation<ins class="diffchange diffchange-inline">"</ins>><table <ins class="diffchange diffchange-inline"> onclick="window.location = 'https://2012.igem.org/Team:Slovenia/SafetyMechanismsMicrocapsuleDegradation';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Microcapsule degradation</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationHepatitisC'><span>Hepatitis C</span></a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationIschaemicHeartDisease'><span>Ischaemic heart disease</span></a></li> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ImplementationImpact'><table <ins class="diffchange diffchange-inline">onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ImplementationImpact';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Impact</span></td><td class="newtable"><img style="margin-right:-86px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/Modeling'><span>Overview</span></a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingPK'><table <ins class="diffchange diffchange-inline">onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingPK';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Pharmacokinetics</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingMethods'><span>Modeling methods</span></a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingMutualRepressorSwitch'><span>Mutual repressor switch</span></a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingPositiveFeedbackLoopSwitch'><span>Positive feedback loop switch</span></a></li> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel'><table <ins class="diffchange diffchange-inline">onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingQuantitativeModel';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Experimental model</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations'><table <ins class="diffchange diffchange-inline">onclick="window.location = 'https://2012.igem.org/Team:Slovenia/ModelingInteractiveSimulations';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Interactive simulations</span></td><td class="newtable"><img style="margin-right:-15px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </ul></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <ul></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/Notebook'><span>Experimental methods</span></a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/NotebookLablog'><table <ins class="diffchange diffchange-inline">onclick="window.location = 'https://2012.igem.org/Team:Slovenia/NotebookLablog';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Lablog</span></td><td class="newtable"><img style="margin-right:-90px;" width="25px" src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png"></img></td></tr></table></a></li> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/NotebookLabSafety'><span>Lab safety</span></a></li> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </ul></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/Team'><span>Team members</span></a></li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TeamAttributions'><span>Attributions</span></a></li></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href='https://2012.igem.org/Team:Slovenia/TeamGallery'><span>Gallery</span></a></li> </div></td></tr>
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Strazkosann
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=288851&oldid=prev
UrbanB at 19:57, 26 October 2012
2012-10-26T19:57:04Z
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UrbanB
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=286294&oldid=prev
Strazkosann at 16:27, 26 October 2012
2012-10-26T16:27:18Z
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Strazkosann
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=286289&oldid=prev
UrbanB at 16:26, 26 October 2012
2012-10-26T16:26:45Z
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Mutual repressor switch</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Mutual repressor switch</h3></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>HEK293T cells were transfected with combinations of plasmids encoding the mutual repressor switch and reporters. Initially we tested the system with the addition of one of the <a href = <del class="diffchange diffchange-inline">“https</del>://2012.igem.org/Team:Slovenia/Parts#<del class="diffchange diffchange-inline">TALeffectors”</del>>TAL repressors</a> expressed constitutively (an internal, constitutive signal) and a luciferase reporter for the opposite TAL. The experiment showed that <b>the second level logic required for the switch works as designed</b> (Figure 5). </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>HEK293T cells were transfected with combinations of plasmids encoding the mutual repressor switch and reporters. Initially we tested the system with the addition of one of the <a href = <ins class="diffchange diffchange-inline">"https</ins>://2012.igem.org/Team:Slovenia/Parts#<ins class="diffchange diffchange-inline">TALeffectors"</ins>>TAL repressors</a> expressed constitutively (an internal, constitutive signal) and a luciferase reporter for the opposite TAL. The experiment showed that <b>the second level logic required for the switch works as designed</b> (Figure 5). </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We also tested the performance of the cellular logic using flow cytometry to measure the number of cells exhibiting fluorescence of each reporter (mCitrine or mNeptune), with different plasmids. The results additionally confirmed the second level logic with constitutively expressed TAL repressors (Figure 6). Unfortunately, we could not detect the mNeptune reporter with the cytometer due to its fluorescence characteristics, but we could detect it using confocal microscopy. </p> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We also tested the performance of the cellular logic using flow cytometry to measure the number of cells exhibiting fluorescence of each reporter (mCitrine or mNeptune), with different plasmids. The results additionally confirmed the second level logic with constitutively expressed TAL repressors (Figure 6). Unfortunately, we could not detect the mNeptune reporter with the cytometer due to its fluorescence characteristics, but we could detect it using confocal microscopy. </p> </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Figure 6. Constitutive TAL repressors drive the switch into the desired state. </b> Numbers of HEK293T cells positive for expression of reporter mCitrine were recorded with flow cytometry. Legend: 1. control - nontransfected cells; 2. cells transfected with p[B]_PCMV_TALA:KRAB_mCitrine (100 ng); 3. cells transfected with <a href=<del class="diffchange diffchange-inline">”http</del>://partsregistry.org/wiki/index.php?title=Part:<del class="diffchange diffchange-inline">BBa_K782083”</del>>p[B]_PCMV_TALA:KRAB_mCitrine</a> and <a href=<del class="diffchange diffchange-inline">”http</del>://partsregistry.org/wiki/index.php?title=Part:<del class="diffchange diffchange-inline">BBa_K782084”</del>>p[A]_PCMV_TALB:KRAB_mNeptune</a> (100 ng each); 4. cells transfected as in 3 with the addition of pPCMV_TALA:KRAB (80 ng); 5. cells transfected as in 3 with the addition of pPCMV_TALB:KRAB (80 ng). The cells were analyzed by flow cytometrythreedays after transfection. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Figure 6. Constitutive TAL repressors drive the switch into the desired state. </b> Numbers of HEK293T cells positive for expression of reporter mCitrine were recorded with flow cytometry. Legend: 1. control - nontransfected cells; 2. cells transfected with p[B]_PCMV_TALA:KRAB_mCitrine (100 ng); 3. cells transfected with <a href=<ins class="diffchange diffchange-inline">"http</ins>://partsregistry.org/wiki/index.php?title=Part:<ins class="diffchange diffchange-inline">BBa_K782083"</ins>>p[B]_PCMV_TALA:KRAB_mCitrine</a> and <a href=<ins class="diffchange diffchange-inline">"http</ins>://partsregistry.org/wiki/index.php?title=Part:<ins class="diffchange diffchange-inline">BBa_K782084"</ins>>p[A]_PCMV_TALB:KRAB_mNeptune</a> (100 ng each); 4. cells transfected as in 3 with the addition of pPCMV_TALA:KRAB (80 ng); 5. cells transfected as in 3 with the addition of pPCMV_TALB:KRAB (80 ng). The cells were analyzed by flow cytometrythreedays after transfection. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h3>Modeling</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h3>Modeling</h3></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>We analyzed the expected performance of the designed TAL repressor-based switch using deterministic as well as stochastic simulation. This analysis (described in detail in the <a href=<del class="diffchange diffchange-inline">”https</del>://2012.igem.org/Team:Slovenia/<del class="diffchange diffchange-inline">Modeling”</del>>Modeling section</a>) revealed that <b>the switch would exhibit bistability only in case of high cooperativity of repressors with the non-linearity coefficient larger than approximately 2</b> (Figures 8 and 9), while the system does not exhibit bistability in case of a non-cooperative repressor binding and transcription (nonlinearity coefficient of 1). Since the crystal structure of a TAL effector bound to DNA indicates that the molecule binds as a monomer to the target DNA (Mak et al., 2012), it is safe to assume that <b>binding of TAL repressors to the binding sites upstream of the promoter is independent and non-cooperative. </b> However the cooperativity of the whole process, including not just binding, but also silencing of the heterochromatin, transcription, and translation, is important for the response. The concentration response of the TAL repressors suggested that the system has a low nonlinearity coefficient. Additionally the simulation indicated that the mutual repressor switch is very <b>sensitive to leaky transcription</b> (Figure 8). </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>We analyzed the expected performance of the designed TAL repressor-based switch using deterministic as well as stochastic simulation. This analysis (described in detail in the <a href=<ins class="diffchange diffchange-inline">"https</ins>://2012.igem.org/Team:Slovenia/<ins class="diffchange diffchange-inline">Modeling"</ins>>Modeling section</a>) revealed that <b>the switch would exhibit bistability only in case of high cooperativity of repressors with the non-linearity coefficient larger than approximately 2</b> (Figures 8 and 9), while the system does not exhibit bistability in case of a non-cooperative repressor binding and transcription (nonlinearity coefficient of 1). Since the crystal structure of a TAL effector bound to DNA indicates that the molecule binds as a monomer to the target DNA (Mak et al., 2012), it is safe to assume that <b>binding of TAL repressors to the binding sites upstream of the promoter is independent and non-cooperative. </b> However the cooperativity of the whole process, including not just binding, but also silencing of the heterochromatin, transcription, and translation, is important for the response. The concentration response of the TAL repressors suggested that the system has a low nonlinearity coefficient. Additionally the simulation indicated that the mutual repressor switch is very <b>sensitive to leaky transcription</b> (Figure 8). </p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b>Figure 8. The mutual repressor switch exhibited no bistability without cooperativity even with a relatively low expression leak (3% of production rates). </b> For a detailed description, see the <a href=<del class="diffchange diffchange-inline">” </del>https://2012.igem.org/Team:Slovenia/<del class="diffchange diffchange-inline">Modeling”</del>>Modeling</a> section. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b>Figure 8. The mutual repressor switch exhibited no bistability without cooperativity even with a relatively low expression leak (3% of production rates). </b> For a detailed description, see the <a href=<ins class="diffchange diffchange-inline">"</ins>https://2012.igem.org/Team:Slovenia/<ins class="diffchange diffchange-inline">Modeling"</ins>>Modeling</a> section. </div></td></tr>
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UrbanB
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=286230&oldid=prev
Strazkosann at 16:22, 26 October 2012
2012-10-26T16:22:03Z
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Strazkosann
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=286206&oldid=prev
Strazkosann at 16:20, 26 October 2012
2012-10-26T16:20:11Z
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Strazkosann
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=286086&oldid=prev
Strazkosann at 16:12, 26 October 2012
2012-10-26T16:12:00Z
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><b>From the experimental and modeling results we can therefore conclude that a simple toggle switch design based on mutual TAL repressors is unstable due to low cooperativity. We thus had to design an <a href=<del class="diffchange diffchange-inline">” </del>https://2012.igem.org/Team:Slovenia/<del class="diffchange diffchange-inline">TheSwitchPositiveFeedbackLoopSwitch”</del>>improved switch</a> to make the switch functional, achieve a higher dynamic range and increased robustness.</b> </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><b>From the experimental and modeling results we can therefore conclude that a simple toggle switch design based on mutual TAL repressors is unstable due to low cooperativity. We thus had to design an <a href=<ins class="diffchange diffchange-inline">" </ins>https://2012.igem.org/Team:Slovenia/<ins class="diffchange diffchange-inline">TheSwitchPositiveFeedbackLoopSwitch"</ins>>improved switch</a> to make the switch functional, achieve a higher dynamic range and increased robustness.</b> </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 style="color:grey;">References</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2 style="color:grey;">References</h2></div></td></tr>
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Strazkosann
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=285955&oldid=prev
Strazkosann at 16:01, 26 October 2012
2012-10-26T16:01:59Z
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Strazkosann
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=283658&oldid=prev
Strazkosann at 12:08, 26 October 2012
2012-10-26T12:08:57Z
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<a href="http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=283658&oldid=283632">Show changes</a>
Strazkosann
http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchMutualRepressorSwitch&diff=283632&oldid=prev
Strazkosann at 12:05, 26 October 2012
2012-10-26T12:05:01Z
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Strazkosann