http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&feed=atom&action=historyTeam:Slovenia/TheSwitchControls - Revision history2024-03-29T14:40:10ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=290531&oldid=prevMiha Jerala at 22:06, 26 October 20122012-10-26T22:06:40Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>In order to remotely control the switch by small moleclar weight inducers, we added an induction system, comprised of two operons that express the appropriate TAL repressor and activator from an inducible promoter (first level plasmids), and one operon that constitutively expresses an inducer-dependent transcription factor (<del class="diffchange diffchange-inline">zero-</del>level plasmid). In the absence of this transcription factor, the inducible promoters act as constitutive promoters. In order to enable switching between two distinct states, two parallel induction systems are required (comprising six operons). We tested the response of the switch when only the first level plasmids of one of the induction systems are added. As shown by fluorescence measurements, the switch shifted into the expected state; plasmids of the erithromycine or tetracycline system shift the switch into state I (mCitrine expression) while plasmids of the pristinamycine system shift the switch into state II (BFP expression). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>In order to remotely control the switch by small moleclar weight inducers, we added an induction system, comprised of two operons that express the appropriate TAL repressor and activator from an inducible promoter (first level plasmids), and one operon that constitutively expresses an inducer-dependent transcription factor (<ins class="diffchange diffchange-inline">second </ins>level plasmid). In the absence of this transcription factor, the inducible promoters act as constitutive promoters. In order to enable switching between two distinct states, two parallel induction systems are required (comprising six operons). We tested the response of the switch when only the first level plasmids of one of the induction systems are added. As shown by fluorescence measurements, the switch shifted into the expected state; plasmids of the erithromycine or tetracycline system shift the switch into state I (mCitrine expression) while plasmids of the pristinamycine system shift the switch into state II (BFP expression). </div></td></tr>
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</table>Miha Jeralahttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=290445&oldid=prevMiha Jerala at 22:01, 26 October 20122012-10-26T22:01:13Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h3>Induction of state I </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h3>Induction of state I <ins class="diffchange diffchange-inline">and state II</ins></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>In order to remotely control the switch by small moleclar weight <del class="diffchange diffchange-inline">inductors</del>, we added an induction system, comprised of two operons that express the appropriate TAL repressor and activator from an inducible promoter (first level plasmids), and one operon that constitutively expresses an inducer-dependent transcription factor (zero-level plasmid). In the absence of this transcription factor, the inducible promoters act as constitutive promoters. In order to enable switching between two distinct states, two parallel induction systems are required (comprising six operons). We tested the response of the switch when only the first level plasmids of one of the induction systems are added. As shown by fluorescence measurements, the switch shifted into the expected state; plasmids of the erithromycine or tetracycline system shift the switch into state I (mCitrine expression) while plasmids of the pristinamycine system shift the switch into state II (BFP expression). </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>In order to remotely control the switch by small moleclar weight <ins class="diffchange diffchange-inline">inducers</ins>, we added an induction system, comprised of two operons that express the appropriate TAL repressor and activator from an inducible promoter (first level plasmids), and one operon that constitutively expresses an inducer-dependent transcription factor (zero-level plasmid). In the absence of this transcription factor, the inducible promoters act as constitutive promoters. In order to enable switching between two distinct states, two parallel induction systems are required (comprising six operons). We tested the response of the switch when only the first level plasmids of one of the induction systems are added. As shown by fluorescence measurements, the switch shifted into the expected state; plasmids of the erithromycine or tetracycline system shift the switch into state I (mCitrine expression) while plasmids of the pristinamycine system shift the switch into state II (BFP expression). </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Next we tested the response of the switch when all of the first level plasmids, but plasmids of only one of the induction systems are present. This should result in the constitutively active promoters on the first level plasmids without their corresponding repressors, and (in the absence of inducer) constitutively repressed promoters on the first level plasmids of the complete induction system. A missing repressor thus in effect simulates a constitutive presence of the corresponding inducer. As expected, the system behaves in the same way as when it is induced. Without the addition of PIP:KRAB, the system expresses BFP (state I) and without the addition of E:KRAB the system expresses mCitrine (state II).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Next we tested the response of the switch when all of the first level plasmids, but <ins class="diffchange diffchange-inline">second level </ins>plasmids <ins class="diffchange diffchange-inline">(E:KRAB and PIP:KRAB) </ins>of only one of the induction systems are present. This should result in the constitutively active promoters on the first level plasmids without their corresponding repressors, and (in the absence of inducer) constitutively repressed promoters on the first level plasmids of the complete induction system. A missing <ins class="diffchange diffchange-inline">inducer-dependant </ins>repressor thus in effect simulates a constitutive presence of the corresponding inducer. As expected, the system behaves in the same way as when it is induced. Without the addition of PIP:KRAB, the system expresses BFP (state I) and without the addition of E:KRAB the system expresses mCitrine (state II).</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>To finally prove the bistability of the complete switch (including the complete induction systems) we transfected the cell with all of the plasmids and left them <del class="diffchange diffchange-inline">either </del>unstimulated <del class="diffchange diffchange-inline">or stimulated them with both of the inducers at once</del>. With microscopy and flow cytometry we observed two populations of cells both expressing only one of the fluorescent reporters, confirming our expectations of bistability as even in the absence of <del class="diffchange diffchange-inline">inducers or presence of both </del>inducers</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>To finally prove the bistability of the complete switch (including the complete induction systems) we transfected the cell with all of the plasmids and left them unstimulated. With microscopy and flow cytometry we observed two populations of cells both expressing only one of the fluorescent reporters, confirming our expectations of bistability as even in the absence of inducers</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>the system stochastically switches to one or the other of the two clearly defined stable states. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>the system stochastically switches to one or the other of the two clearly defined stable states. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The switch coupled with both induction systems - both inducers present simultaneously </h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>The switch coupled with both induction systems - both inducers present simultaneously </h3></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br/><p>Some of the judges at <del class="diffchange diffchange-inline">iGEM </del>were curious what would happen if the system is stimulated with both inducers <del class="diffchange diffchange-inline">symultaneously</del>. <del class="diffchange diffchange-inline">This type of </del>combination of both signals <del class="diffchange diffchange-inline">results </del>in <del class="diffchange diffchange-inline">the </del>electronic circuits <del class="diffchange diffchange-inline">as </del>an undefined state. Experimental results for our switch suggest that both reporters are effectively repressed, as we determined that transcription <del class="diffchange diffchange-inline">factor </del>with KRAB is able to <del class="diffchange diffchange-inline">repress </del>the effect of a transcription <del class="diffchange diffchange-inline">factor comprisong VP16 </del>activator. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br/><p>Some of the judges at <ins class="diffchange diffchange-inline">the European Jamboree </ins>were curious <ins class="diffchange diffchange-inline">as to </ins>what would happen if the system is stimulated with both inducers <ins class="diffchange diffchange-inline">simultaneously</ins>. <ins class="diffchange diffchange-inline">A </ins>combination of both signals in electronic circuits <ins class="diffchange diffchange-inline">results in </ins>an undefined state. Experimental results for our switch suggest that both reporters are effectively repressed, as we determined that <ins class="diffchange diffchange-inline">a </ins>transcription <ins class="diffchange diffchange-inline">repressor </ins>with <ins class="diffchange diffchange-inline">the </ins>KRAB <ins class="diffchange diffchange-inline">domain </ins>is able to <ins class="diffchange diffchange-inline">overpower </ins>the effect of a transcription activator <ins class="diffchange diffchange-inline">with the VP16 domain</ins>. </div></td></tr>
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</table>Miha Jeralahttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=289767&oldid=prevStrazkosann at 21:09, 26 October 20122012-10-26T21:09:57Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><del class="diffchange diffchange-inline">In order to investigate the leakage of minimal promoters in the </del>switch, we <del class="diffchange diffchange-inline">performed two controls that included </del>the plasmids <del class="diffchange diffchange-inline">of one or </del>the <del class="diffchange diffchange-inline">other state of the switch but no </del>induction system. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">To determine whether our improved </ins>switch <ins class="diffchange diffchange-inline">is bistable</ins>, we <ins class="diffchange diffchange-inline">transfected HEK293T cells with all four constructs comprising </ins>the <ins class="diffchange diffchange-inline">bistable switch with a positive feedback loop, without </ins>plasmids <ins class="diffchange diffchange-inline">comprising </ins>the induction system. We observed <ins class="diffchange diffchange-inline">fluorescence of both reporters as cells randomly shift </ins>to <ins class="diffchange diffchange-inline">one of </ins>the <ins class="diffchange diffchange-inline">two possible states [A]. We could observe that each reporter was expressed </ins>in <ins class="diffchange diffchange-inline">different </ins>cells <ins class="diffchange diffchange-inline">as shown in the overlay and no fluorescence colocalization was </ins>determined <ins class="diffchange diffchange-inline">[B]. This clearly demonstrates bistability </ins>of the positive feedback loop <ins class="diffchange diffchange-inline">in contrast to </ins>the <ins class="diffchange diffchange-inline">simple mutual repressor switch which demonstrated high degree </ins>of <ins class="diffchange diffchange-inline">fluorescence overlap (Figure 7 </ins>of the <ins class="diffchange diffchange-inline">Mutual </ins>repressor <ins class="diffchange diffchange-inline">section)</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We observed <del class="diffchange diffchange-inline">flourescence corresponding </del>to the <del class="diffchange diffchange-inline">transfected plasmids </del>in <del class="diffchange diffchange-inline">few </del>cells<del class="diffchange diffchange-inline">, indicating some promoter leakage. Even though we </del>determined <del class="diffchange diffchange-inline">a minimal leakage </del>of <del class="diffchange diffchange-inline">the minimal promoter this minimal promoter leakage can nevertheless initiate transcription of the activator which in turn triggers </del>the positive feedback loop <del class="diffchange diffchange-inline">and causes further activation of </del>the <del class="diffchange diffchange-inline">system and expression </del>of <del class="diffchange diffchange-inline">the reporter. Leakage </del>of the <del class="diffchange diffchange-inline">minimal promoter and amplification of gene expression were detected for both states of the switch which also indicates that there is no difference if the fluorescent reporter protein is linked to an activator or to a </del>repressor.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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</table>Strazkosannhttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=289545&oldid=prevStrazkosann at 20:53, 26 October 20122012-10-26T20:53:26Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><body></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><body></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><a name="topofthepage" style="background-color:transparent;"></a></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div id="banner"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div id="banner"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a style="position:absolute; top:0px; left:490px;" href="https://2012.igem.org/Main_Page"><b></b></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a style="position:absolute; top:0px; left:490px;" href="https://2012.igem.org/Main_Page"><b></b></a></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 352:</td>
<td colspan="2" class="diff-lineno">Line 365:</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/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>
<tr><td colspan="2" class="diff-lineno">Line 361:</td>
<td colspan="2" class="diff-lineno">Line 374:</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/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>
<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>
<tr><td colspan="2" class="diff-lineno">Line 369:</td>
<td colspan="2" class="diff-lineno">Line 382:</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/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>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </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 colspan="2" class="diff-lineno">Line 377:</td>
<td colspan="2" class="diff-lineno">Line 390:</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><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>
<tr><td colspan="2" class="diff-lineno">Line 393:</td>
<td colspan="2" class="diff-lineno">Line 406:</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><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>
<tr><td colspan="2" class="diff-lineno">Line 416:</td>
<td colspan="2" class="diff-lineno">Line 429:</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/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>
<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/TeamCollaborations'><table class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" 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/TeamCollaborations'><table <ins class="diffchange diffchange-inline"> onclick="window.location = 'https://2012.igem.org/Team:Slovenia/TeamCollaborations';" </ins>class="newtable"><tr class="newtable"><td class="newtable"><span>Collaborations</span></td><td class="newtable"><img style="margin-right:-20px;" 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/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>
<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/TeamSponsors'><span>Sponsors</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/TeamSponsors'><span>Sponsors</span></a></li> </div></td></tr>
</table>Strazkosannhttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=289129&oldid=prevUrbanB at 20:20, 26 October 20122012-10-26T20:20:36Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><p>For the switch to function properly, all of TAL effectors need to exhibit high specificity and orthogonality. We tested different combinations of TAL operons and TAL activators (Figure) to control for any cross-reactivity and binding specificity. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">When a reporter under the TAL operon was cotransfected with its matching TAL activator, fluorescence was detected. When the transfected reported plasmid‘s operon did not match the cotransfected TAL activator, no flourescence was detected even if it contained 10 copies of two other TAL binding sites. This demonstrates that TAL regulators bind and exert activity specifically at their binding sites and <b>high degree of orthogonality. </b></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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</table>UrbanBhttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=288826&oldid=prevUrbanB at 19:56, 26 October 20122012-10-26T19:56:09Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><hr color="gray"/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><hr color="gray"/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We prepared a switch with inducible systems comprising CL1-PEST degradation tags (see <del class="diffchange diffchange-inline">figure</del>/<del class="diffchange diffchange-inline">control 1</del>), which should allow faster switching between states. We tested how these improved induction systems induce one state of the switch or the other. We also tested if the switch retains the stable state if the inducer is removed.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We prepared a switch with inducible systems comprising CL1-PEST degradation tags (<ins class="diffchange diffchange-inline"><a href="https://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls#blava"></ins>see <ins class="diffchange diffchange-inline">above<</ins>/<ins class="diffchange diffchange-inline">a></ins>), which should allow faster switching between states. We tested how these improved induction systems induce one state of the switch or the other. We also tested if the switch retains the stable state if the inducer is removed.</div></td></tr>
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</table>UrbanBhttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=288807&oldid=prevDusanv at 19:55, 26 October 20122012-10-26T19:55:12Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><br/></del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Inducible TAL regulators</h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3>Inducible TAL regulators</h3></div></td></tr>
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</table>Dusanvhttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=288798&oldid=prevDusanv at 19:54, 26 October 20122012-10-26T19:54:46Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next: <a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'>Safety mechanisms >></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Next: <a href='https://2012.igem.org/Team:Slovenia/SafetyMechanisms'>Safety mechanisms >></a></div></td></tr>
</table>Dusanvhttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=288587&oldid=prevUrbanB at 19:40, 26 October 20122012-10-26T19:40:04Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>TAL activator specifically recognizes its corresponding DNA-binding site. </b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>TAL activator specifically recognizes its corresponding DNA-binding site. </b></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>HEK293T cells were cotransfected with transfection control PCMV_ mCherry (10 ng), PCMV_TALA:VP16 <del class="diffchange diffchange-inline">[</del>a,b<del class="diffchange diffchange-inline">] </del>or PCMV_TALB:VP16 (c) and reporter [AB]_PCMV_mCit (a), [CB]_PCMV_mCit (b), or [AC]_PCMV_mCit (all 10 ng) (b). Fluorescence was detected only in cells cotransfected with the specific TAL effector and its corresponding DNA-binding site (a), while cross-reactivity with multiple copies of other binding sites was not observed (b and c).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>HEK293T cells were cotransfected with transfection control PCMV_ mCherry (10 ng), PCMV_TALA:VP16 <ins class="diffchange diffchange-inline">(</ins>a,b<ins class="diffchange diffchange-inline">) </ins>or PCMV_TALB:VP16 (c) and reporter [AB]_PCMV_mCit (a), [CB]_PCMV_mCit (b), or [AC]_PCMV_mCit (all 10 ng) (b). Fluorescence was detected only in cells cotransfected with the specific TAL effector and its corresponding DNA-binding site (a), while cross-reactivity with multiple copies of other binding sites was not observed (b and c).</div></td></tr>
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</table>UrbanBhttp://2012.igem.org/wiki/index.php?title=Team:Slovenia/TheSwitchControls&diff=288563&oldid=prevUrbanB: Undo revision 288554 by UrbanB (talk)2012-10-26T19:38:17Z<p>Undo revision 288554 by <a href="/Special:Contributions/UrbanB" title="Special:Contributions/UrbanB">UrbanB</a> (<a href="/wiki/index.php?title=User_talk:UrbanB&action=edit&redlink=1" class="new" title="User talk:UrbanB (page does not exist)">talk</a>)</p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>TAL activator specifically recognizes its corresponding DNA-binding site. </b></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>TAL activator specifically recognizes its corresponding DNA-binding site. </b></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>HEK293T cells were cotransfected with transfection control PCMV_ mCherry (10 ng), PCMV_TALA:VP16 <del class="diffchange diffchange-inline">(</del>a,b<del class="diffchange diffchange-inline">) </del>or PCMV_TALB:VP16 (c) and reporter [AB]_PCMV_mCit (a), [CB]_PCMV_mCit (b), or [AC]_PCMV_mCit (all 10 ng) (b). Fluorescence was detected only in cells cotransfected with the specific TAL effector and its corresponding DNA-binding site (a), while cross-reactivity with multiple copies of other binding sites was not observed (b and c).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>HEK293T cells were cotransfected with transfection control PCMV_ mCherry (10 ng), PCMV_TALA:VP16 <ins class="diffchange diffchange-inline">[</ins>a,b<ins class="diffchange diffchange-inline">] </ins>or PCMV_TALB:VP16 (c) and reporter [AB]_PCMV_mCit (a), [CB]_PCMV_mCit (b), or [AC]_PCMV_mCit (all 10 ng) (b). Fluorescence was detected only in cells cotransfected with the specific TAL effector and its corresponding DNA-binding site (a), while cross-reactivity with multiple copies of other binding sites was not observed (b and c).</div></td></tr>
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</table>UrbanB