http://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&feed=atom&action=historyTeam:Edinburgh/Bacterial/Red light sensor - Revision history2024-03-28T11:02:44ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=205503&oldid=prevDonal at 02:27, 28 October 20102010-10-28T02:27:30Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future <del class="diffchange diffchange-inline">work</del></a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future"><ins class="diffchange diffchange-inline">the </ins>future</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://2010.igem.org/Team:Edinburgh/Project/References">references</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</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://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future <del class="diffchange diffchange-inline">work</del></a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future"><ins class="diffchange diffchange-inline">the </ins>future</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://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li></div></td></tr>
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</table>Donalhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=203115&oldid=prevJRWK at 00:56, 28 October 20102010-10-28T00:56:45Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Overview" id="Overview"></a><h2>Overview: The red light sensor</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Overview" id="Overview"></a><h2>Overview: The red light sensor</h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The red light sensor was first engineered by <a href="http://partsregistry.org/Coliroid">UT Austin in iGEM 2004</a>. In this project, we will try to use it as one of the photo-activated sensors for our <a href="https://2010.igem.org/Team:Edinburgh/Bacterial">FORTH framework</a>. The maximum response of the sensor is at a wavelength of 660nm.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The red light sensor was first <ins class="diffchange diffchange-inline"><b></ins>engineered<ins class="diffchange diffchange-inline"></b> </ins>by <a href="http://partsregistry.org/Coliroid">UT Austin in iGEM 2004</a>. In this project, we will try to use it as one of the photo-activated sensors for our <a href="https://2010.igem.org/Team:Edinburgh/Bacterial">FORTH framework</a>. The maximum response of the sensor is at a wavelength of 660nm.</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>The red-light sensor (Cph8) contains three parts:</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The red-light sensor (Cph8) contains three parts:</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Strategy" id="Strategy"></a><h2>Strategy</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Strategy" id="Strategy"></a><h2>Strategy</h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Our original plan was to revive <a href="http://partsregistry.org/Coliroid">UT Austin 2004</a>'s BioBricks <a href="http://partsregistry.org/Part:BBa_I15008">BBa_I15008</a>, <a href="http://partsregistry.org/Part:BBa_I15009">BBa_I15009</a>, and <a href="http://partsregistry.org/Part:BBa_I15010">BBa_I15010</a> from the provided Registry plates. Once revived, we aimed to combine them into a single red light sensing system, and transform cells with for characterisation of the system and for analysis of their compatibility with the mutated <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red luciferases</a>.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Our original plan was to <ins class="diffchange diffchange-inline"><b></ins>revive<ins class="diffchange diffchange-inline"></b> </ins><a href="http://partsregistry.org/Coliroid">UT Austin 2004</a>'s BioBricks <a href="http://partsregistry.org/Part:BBa_I15008">BBa_I15008</a>, <a href="http://partsregistry.org/Part:BBa_I15009">BBa_I15009</a>, and <a href="http://partsregistry.org/Part:BBa_I15010">BBa_I15010</a> from the provided Registry plates. Once revived, we aimed to combine them into a single red light sensing system, and transform cells with for <ins class="diffchange diffchange-inline"><b></ins>characterisation<ins class="diffchange diffchange-inline"></b> </ins>of the system and for <ins class="diffchange diffchange-inline"><b></ins>analysis<ins class="diffchange diffchange-inline"></b> </ins>of their compatibility with the mutated <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red luciferases</a>.</p><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Problems" id="Problems"></a><h2>Problems</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Problems" id="Problems"></a><h2>Problems</h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The composite construct of all three BioBricks would not transform, and transformations of the individual BioBricks also failed. We attempted to amplify products out of the BioBrick to see if they were actually there, but the only component that we were able to recover was the sensing component, Cph8.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The composite construct of all three BioBricks would not transform, and transformations of the individual BioBricks also <ins class="diffchange diffchange-inline"><b></ins>failed<ins class="diffchange diffchange-inline"></b></ins>. We attempted to amplify products out of the BioBrick to see if they were actually there, but the only component that we were able to <ins class="diffchange diffchange-inline"><b></ins>recover<ins class="diffchange diffchange-inline"></b> </ins>was the sensing component, Cph8.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Thus, we retrieved transformants from the kanamycin-resistant version of <a href="http://partsregistry.org/Part:BBa_K098010">BBa_K098010</a>, the HO-pcyA fusion deposited by <a href="https://2008.igem.org/Team:Harvard">Harvard 2008</a>, to supplement this and to complete our red light sensor.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Thus, we retrieved transformants from the kanamycin-resistant version of <a href="http://partsregistry.org/Part:BBa_K098010">BBa_K098010</a>, the HO-pcyA fusion deposited by <a href="https://2008.igem.org/Team:Harvard">Harvard 2008</a>, to <ins class="diffchange diffchange-inline"><b></ins>supplement<ins class="diffchange diffchange-inline"></b> </ins>this and to complete our red light sensor.</p><br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The red light sensor has seen frequent use throughout the history of iGEM, beginning with the original coliroid parts by <a href="http://partsregistry.org/Coliroid">UT Austin 2004</a> to their adaptation by <a href="https://2008.igem.org/Team:Harvard">Harvard 2008</a>. We have updated and adapted their parts to the pSB1C3 chassis along with a number of different reporter systems for characterisation.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The red light sensor has seen frequent use throughout the history of iGEM, beginning with the original coliroid parts by <a href="http://partsregistry.org/Coliroid">UT Austin 2004</a> to their <ins class="diffchange diffchange-inline"><b></ins>adaptation<ins class="diffchange diffchange-inline"></b> </ins>by <a href="https://2008.igem.org/Team:Harvard">Harvard 2008</a>. We have updated and adapted their parts to the pSB1C3 chassis along with a number of different reporter systems for characterisation.</p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="http://partsregistry.org/Part:BBa_K322122">BBa_K322122</a>: phycocyanobilin synthesis operon (Harvard 2008's <a href="http://partsregistry.org/Part:BBa_K098010">BBa_K098010</a> in pSB1C3).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="http://partsregistry.org/Part:BBa_K322122">BBa_K322122</a>: phycocyanobilin synthesis operon (Harvard 2008's <a href="http://partsregistry.org/Part:BBa_K098010">BBa_K098010</a> in pSB1C3).</p></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=192335&oldid=prevJRWK at 18:52, 27 October 20102010-10-27T18:52:58Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 18:52, 27 October 2010</td>
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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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>background-image:url("https://static.igem.org/mediawiki/2010/a/a8/Ed10-LargePaper.jpg");</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>background-image:url("https://static.igem.org/mediawiki/2010/a/a8/Ed10-LargePaper.jpg");</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In the absence of red light, phosphorylated OmpR activates EnvZ, which in turn promotes transcription from the <i>ompC</i> promoter and represses transcription from the <i>ompF</i> promoter, thus leading to the expression of LacZ (in this case under the regulation of the aforementioned promoter). This catalyses the formation of a black precipitate from S-gal(3,4-cyclohexenoesculetin-β-D-galactopyranoside).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In the absence of red light, phosphorylated OmpR activates EnvZ, which in turn promotes transcription from the <i>ompC</i> promoter and represses transcription from the <i>ompF</i> promoter, thus leading to the expression of LacZ (in this case under the regulation of the aforementioned promoter). This catalyses the formation of a black precipitate from S-gal(3,4-cyclohexenoesculetin-β-D-galactopyranoside).</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>When exposed to red light, an isomerization in the Cph1 and a structure change in the phycocyanobilin (PCB) part of the photoreceptor inactivates the histidine kinase acitity of EnvZ (<a href="https://static.igem.org/mediawiki/2010/8/8c/Ed10-RedSensor.png">Figure 1</a>).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>When exposed to red light, an isomerization in the Cph1 and a structure change in the phycocyanobilin (PCB) part of the photoreceptor inactivates the histidine kinase acitity of EnvZ (<a href="https://static.igem.org/mediawiki/2010/8/8c/Ed10-RedSensor.png">Figure 1</a>)<ins class="diffchange diffchange-inline">. This then cascades down the OmpR pathway as shown in <a href="https://static.igem.org/mediawiki/2010/thumb/0/08/Ed10-Cph8.jpg/508px-Ed10-Cph8.jpg">Figure 2</a></ins>.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/8/8c/Ed10-RedSensor.png" width="600px"></p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/8/8c/Ed10-RedSensor.png" width="600px"></p><br></div></td></tr>
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<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;"></ins></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;"><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/thumb/0/08/Ed10-Cph8.jpg/508px-Ed10-Cph8.jpg"></p><br><p><b>Figure 2:</b> Another view of the red light sensor.</p><br><br></center></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Problems" id="Problems"></a><h2>Problems</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Problems" id="Problems"></a><h2>Problems</h2></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="http://partsregistry.org/Part:BBa_K322127">BBa_K322127</a>: phycocyanobilin synthesis genes with <i>cph8</i>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="http://partsregistry.org/Part:BBa_K322127">BBa_K322127</a>: phycocyanobilin synthesis genes with <i>cph8</i>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="http://partsregistry.org/Part:BBa_K322128">BBa_K322128</a>: phycocyanobilin synthesis genes with <i>cph8</i> and EYFP reporter system.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="http://partsregistry.org/Part:BBa_K322128">BBa_K322128</a>: phycocyanobilin synthesis genes with <i>cph8</i> and EYFP reporter system.</p><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure <del class="diffchange diffchange-inline">2</del></a>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure <ins class="diffchange diffchange-inline">3</ins></a>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Further characterisation of the RLS was attempted using the <del class="diffchange diffchange-inline">Beta-Galactosidase Assay (A Better Miller) from OpenWetWare </del>http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) and preliminary data was recorded and calculated as shown in Figure <del class="diffchange diffchange-inline">3</del>. The cultures grown were as follows:</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png" width="600px"></p><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p><b>Figure 3:</b> Characterisation data for the red light sensor.</p><br><br></center></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins>Further characterisation of the RLS was attempted using the <ins class="diffchange diffchange-inline"><a href="</ins>http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller)<ins class="diffchange diffchange-inline">">Beta-Galactosidase Assay (A Better Miller) from OpenWetWare</a> </ins>and preliminary data was recorded and calculated as shown in <ins class="diffchange diffchange-inline"><a href="</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">https://static.igem.org/mediawiki/2010/1/16/Ed10-RedLightSensCharData2.jpg"></ins>Figure <ins class="diffchange diffchange-inline">4</a></ins>. The cultures grown were as follows:</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> and the </del>conditions were Cml 40 <del class="diffchange diffchange-inline">& </del>IPTG 90 in similar ONs to those used in the YFP characterisation.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p>The </ins>conditions were Cml 40 <ins class="diffchange diffchange-inline">and </ins>IPTG 90 in similar ONs to those used in the YFP characterisation.<ins class="diffchange diffchange-inline"></p><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><<del class="diffchange diffchange-inline">center</del>><<del class="diffchange diffchange-inline">br</del>><br><p><del class="diffchange diffchange-inline"><img src</del>=<del class="diffchange diffchange-inline">"https://static</del>.<del class="diffchange diffchange-inline">igem.org</del>/<del class="diffchange diffchange-inline">mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png" width="600px"</del>><<del class="diffchange diffchange-inline">/</del>p><<del class="diffchange diffchange-inline">br</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">h3</ins>><ins class="diffchange diffchange-inline">YFP RLS Characterisation</ins><<ins class="diffchange diffchange-inline">/h3</ins>><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><<del class="diffchange diffchange-inline">b</del>><del class="diffchange diffchange-inline">Figure 2:</del></<del class="diffchange diffchange-inline">b</del>> <del class="diffchange diffchange-inline">Characterisation data for </del>the <del class="diffchange diffchange-inline">red </del>light <del class="diffchange diffchange-inline">sensor</del>.</p><<del class="diffchange diffchange-inline">br</del>><<del class="diffchange diffchange-inline">br</del>></<del class="diffchange diffchange-inline">center</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">Red light </ins>= <ins class="diffchange diffchange-inline">less yellow colour fluorescence</ins>.<ins class="diffchange diffchange-inline"><</ins>/<ins class="diffchange diffchange-inline">p</ins>></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">No Red light = more yellow colour fluorescence.</ins><<ins class="diffchange diffchange-inline">/p</ins>></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">This assay shows the fluorescence per unit of estimated biomass for a number of different times of incubation. We can see that the controls for YFP in both the JM109 and </ins><<ins class="diffchange diffchange-inline">i</ins>><ins class="diffchange diffchange-inline">envZ</ins></<ins class="diffchange diffchange-inline">i</ins>> <ins class="diffchange diffchange-inline">mutant strains show reasonable paired results in both </ins>the light <ins class="diffchange diffchange-inline">and the dark. This means that the controls work as expected, however the large difference between the JM109 and the <i>envZ</i> control pairs implies that most of the result seen in JM109 is due to background <i>envZ</i> activity</ins>.</p></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">p</ins>><ins class="diffchange diffchange-inline">The rest of the results however are of limited usefulness beyond the testing of our assay procedure and the benchmark they represent of the RLS system which we can use to further improve our characterisation assays. The reasons for the affect seen are similar to those listed above in the </ins><<ins class="diffchange diffchange-inline">i</ins>><ins class="diffchange diffchange-inline">lacZ</ins></<ins class="diffchange diffchange-inline">i> RLS characterisation, listed below.</p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br</ins>></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></div></td></tr>
<tr><td colspan="2" class="diff-lineno">Line 249:</td>
<td colspan="2" class="diff-lineno">Line 274:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>https://static.igem.org/mediawiki/2010/1/16/Ed10-RedLightSensCharData2.jpg" width="600px"></p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>https://static.igem.org/mediawiki/2010/1/16/Ed10-RedLightSensCharData2.jpg" width="600px"></p><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><b>Figure <del class="diffchange diffchange-inline">3</del>:</b> Further characterisation data for the red light sensor.</p><br><br></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><b>Figure <ins class="diffchange diffchange-inline">4</ins>:</b> Further characterisation data for the red light sensor.</p><br><br></center></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </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"><p><a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 3</a> shows further characterisation data for the red light sensor.</p></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> </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"><p>Analysis.</p><br></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> </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">YFP RLS Characterisation</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> </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">Red light = less yellow colour fluorescence</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">No Red light = more yellow colour fluorescence</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">This assay shows the fluorescence per unit of estimated biomass for a number of different times of incubation. We can see that the controls for YFP in both the JM109 & envZ mutant strains show reasonable paired results for both Light and Dark. This means that the controls work as expected, however the large difference between the JM109 & the envZ control pairs implies that most of the result seen in JM109 is due to background envZ activity. </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">The rest of the results however are of limited usefulness beyond the testing of our assay procedure and the benchmark they represent of the RLS system which we can use to further improve our characterisation assays. The reasons for the affect seen are similar to those listed above in the lacZ RLS characterisation, listed below.</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> </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: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<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;">lacZ RLS Characterisation</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">Red light = less yellow colour ONPG</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h3></ins>lacZ RLS <ins class="diffchange diffchange-inline">Characterisation<</ins>/<ins class="diffchange diffchange-inline">h3><br></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">No Red light = more yellow colour ONPG</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">This characterisation data was acquired using a β-Galactosidase substrate o-nitrophenyl-β-D-galactoside (ONPG) which is cleaved to form a yellow dye that can be easily visualised and absorbance measurements can be taken. This can be used as a direct measure of </del>lacZ <del class="diffchange diffchange-inline">activity as lacZ encodes for β-Galactosidase and when ONPG is in excess the production of the visible yellow dye can be measured over time to shown lacZ activation and control via promoter systems.</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">The absorbance at 420nm was divided by the time taken for the colour of o-nitrophenyl to become visible and was used to calculate a rate of expression.</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">This preliminary Miller-like assay shows limited useful characterisation date of the Red Light Sensor (</del>RLS<del class="diffchange diffchange-inline">) system, and is a good start towards accurate characterisation of the system, which we hope to continue now that a preliminary methodology has been found and tested.</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">The envZ – RLS.lacZ.YFP (Light </del>/ <del class="diffchange diffchange-inline">Dark) cultures did not produce Miller-like results, as was expected and have such not been included in the graphs, they were however run as a negative control to prove that any effects seen were not due to an artefact from the envZ mutant.</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">Though the data is not entirely useful we believe it may be due to a number of potential causes – such as the cofactor not being expressed properly, weak light intensity unable to activate the RLS system, or insufficient expression of the system as a whole - all of which we could test for given sufficient time, allowing us to improve our experimental technique to account for accordingly. We will endeavour to do so and bring further results to the Jamboree.</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: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></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;"><p>Red light = less yellow colour ONPG</p></ins></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;"><p>No Red light = more yellow colour ONPG</p></ins></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;"><p>This characterisation data was acquired using a β-Galactosidase substrate o-nitrophenyl-β-D-galactoside (ONPG) which is cleaved to form a yellow dye that can be easily visualised and absorbance measurements can be taken. This can be used as a direct measure of <i>lacZ</i> activity as <i>lacZ</i> encodes for β-Galactosidase and when ONPG is in excess the production of the visible yellow dye can be measured over time to shown <i>lacZ</i> activation and control via promoter systems.</p></ins></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;"><p>The absorbance at 420nm was divided by the time taken for the colour of o-nitrophenyl to become visible and was used to calculate a rate of expression.</p></ins></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;"><p>This preliminary Miller-like assay shows limited useful characterisation date of the red light sensor (RLS) system, and is a good start towards accurate characterisation of the system, which we hope to continue now that a preliminary methodology has been found and tested.</p></ins></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;"><p>The <i>envZ</i> – RLS.lacZ.YFP (light / dark) cultures did not produce Miller-like results, as was expected and have such not been included in the graphs, they were however run as a negative control to prove that any effects seen were not due to an artefact from the <i>envZ</i> mutant.</p></ins></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;"><p>Though the data is not entirely useful we believe it may be due to a number of potential causes – such as the cofactor not being expressed properly, weak light intensity unable to activate the RLS system, or insufficient expression of the system as a whole - all of which we could test for given sufficient time, allowing us to improve our experimental technique to account for accordingly. We will endeavour to do so and bring further results to the Jamboree.</p><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=190972&oldid=prevRichardPH at 18:10, 27 October 20102010-10-27T18:10:20Z<p></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The absorbance at 420nm was divided by the time taken for the colour of o-nitrophenyl to become visible and was used to calculate a rate of expression.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The absorbance at 420nm was divided by the time taken for the colour of o-nitrophenyl to become visible and was used to calculate a rate of expression.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This preliminary Miller-like assay shows limited useful characterisation date of the Red Light Sensor (RLS) system, and is a good start towards accurate characterisation of the system, which we hope to continue now that a preliminary methodology has been found and tested.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This preliminary Miller-like assay shows limited useful characterisation date of the Red Light Sensor (RLS) system, and is a good start towards accurate characterisation of the system, which we hope to continue now that a preliminary methodology has been found and tested.</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;">The envZ – RLS.lacZ.YFP (Light / Dark) cultures did not produce Miller-like results, as was expected and have such not been included in the graphs, they were however run as a negative control to prove that any effects seen were not due to an artefact from the envZ mutant.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Though the data is not entirely useful we believe it may be due to a number of potential causes – such as the cofactor not being expressed properly, weak light intensity unable to activate the RLS system, or insufficient expression of the system as a whole - all of which we could test for given sufficient time, allowing us to improve our experimental technique to account for accordingly. We will endeavour to do so and bring further results to the Jamboree.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Though the data is not entirely useful we believe it may be due to a number of potential causes – such as the cofactor not being expressed properly, weak light intensity unable to activate the RLS system, or insufficient expression of the system as a whole - all of which we could test for given sufficient time, allowing us to improve our experimental technique to account for accordingly. We will endeavour to do so and bring further results to the Jamboree.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>RichardPHhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=190873&oldid=prevRichardPH at 18:07, 27 October 20102010-10-27T18:07:19Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 2</a>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 2</a>.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Further characterisation of the RLS was attempted using the Beta-Galactosidase Assay (A Better Miller) from OpenWetWare http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) and preliminary data was recorded and calculated as shown in Figure 3.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Further characterisation of the RLS was attempted using the Beta-Galactosidase Assay (A Better Miller) from OpenWetWare http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) and preliminary data was recorded and calculated as shown in Figure 3<ins class="diffchange diffchange-inline">. The cultures grown were as follows:</p></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><ul></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> <li>JM109 – RLS.lacZ.YFP (Light / Dark)</li></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> <li>envZ – RLS.lacZ.YFP (Light / Dark)</li></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> <li>JM109 – lacZ Control (Light / Dark)</li></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> <li>JM109 – RLS.lacZ (Light / Dark)</li></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"></ul></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> and the conditions were Cml 40 & IPTG 90 in similar ONs to those used in the YFP characterisation</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png" width="600px"></p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png" width="600px"></p><br></div></td></tr>
</table>RichardPHhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=190773&oldid=prevRichardPH at 18:04, 27 October 20102010-10-27T18:04:12Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 2</a>.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 2</a>.</p></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;"></ins></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;">Further characterisation of the RLS was attempted using the Beta-Galactosidase Assay (A Better Miller) from OpenWetWare http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) and preliminary data was recorded and calculated as shown in Figure 3.</ins></div></td></tr>
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</table>RichardPHhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=190537&oldid=prevRichardPH at 17:57, 27 October 20102010-10-27T17:57:33Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Analysis.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Analysis.</p><br></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;"></ins></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;">YFP RLS Characterisation</ins></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;"></ins></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;">Red light = less yellow colour fluorescence</ins></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;">No Red light = more yellow colour fluorescence</ins></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;">This assay shows the fluorescence per unit of estimated biomass for a number of different times of incubation. We can see that the controls for YFP in both the JM109 & envZ mutant strains show reasonable paired results for both Light and Dark. This means that the controls work as expected, however the large difference between the JM109 & the envZ control pairs implies that most of the result seen in JM109 is due to background envZ activity. </ins></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;">The rest of the results however are of limited usefulness beyond the testing of our assay procedure and the benchmark they represent of the RLS system which we can use to further improve our characterisation assays. The reasons for the affect seen are similar to those listed above in the lacZ RLS characterisation, listed below.</ins></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;"></ins></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;"></ins></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;">lacZ RLS Characterisation</ins></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;"></ins></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;">Red light = less yellow colour ONPG</ins></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;">No Red light = more yellow colour ONPG</ins></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;">This characterisation data was acquired using a β-Galactosidase substrate o-nitrophenyl-β-D-galactoside (ONPG) which is cleaved to form a yellow dye that can be easily visualised and absorbance measurements can be taken. This can be used as a direct measure of lacZ activity as lacZ encodes for β-Galactosidase and when ONPG is in excess the production of the visible yellow dye can be measured over time to shown lacZ activation and control via promoter systems.</ins></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;">The absorbance at 420nm was divided by the time taken for the colour of o-nitrophenyl to become visible and was used to calculate a rate of expression.</ins></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;">This preliminary Miller-like assay shows limited useful characterisation date of the Red Light Sensor (RLS) system, and is a good start towards accurate characterisation of the system, which we hope to continue now that a preliminary methodology has been found and tested.</ins></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;">Though the data is not entirely useful we believe it may be due to a number of potential causes – such as the cofactor not being expressed properly, weak light intensity unable to activate the RLS system, or insufficient expression of the system as a whole - all of which we could test for given sufficient time, allowing us to improve our experimental technique to account for accordingly. We will endeavour to do so and bring further results to the Jamboree.</ins></div></td></tr>
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</table>RichardPHhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=190272&oldid=prevJRWK at 17:50, 27 October 20102010-10-27T17:50:28Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>https://static.igem.org/mediawiki/2010/1/16/Ed10-RedLightSensCharData2.jpg" width="<del class="diffchange diffchange-inline">450px</del>"></p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>https://static.igem.org/mediawiki/2010/1/16/Ed10-RedLightSensCharData2.jpg" width="<ins class="diffchange diffchange-inline">600px</ins>"></p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 3:</b> Further characterisation data for the red light sensor.</p><br><br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 3:</b> Further characterisation data for the red light sensor.</p><br><br></center></div></td></tr>
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</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=188164&oldid=prevJRWK at 16:43, 27 October 20102010-10-27T16:43:24Z<p></p>
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<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;">https://static.igem.org/mediawiki/2010/1/16/Ed10-RedLightSensCharData2.jpg" width="450px"></p><br></ins></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;"><p><b>Figure 3:</b> Further characterisation data for the red light sensor.</p><br><br></center></ins></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;"></ins></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;"><p><a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 3</a> shows further characterisation data for the red light sensor.</p></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Analysis.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Analysis.</p><br></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_sensor&diff=184278&oldid=prevJRWK at 14:32, 27 October 20102010-10-27T14:32:51Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Overview" id="Overview"></a><h2>Overview: The red light sensor</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="Overview" id="Overview"></a><h2>Overview: The red light sensor</h2></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The red light sensor was first engineered by <a href="http://partsregistry.org/Coliroid">UT Austin in iGEM 2004</a>. In this project, we will try to use it as one of the photo-activated sensors <del class="diffchange diffchange-inline">in the repressilator network</del>. The maximum response of the sensor is at a wavelength of 660nm.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The red light sensor was first engineered by <a href="http://partsregistry.org/Coliroid">UT Austin in iGEM 2004</a>. In this project, we will try to use it as one of the photo-activated sensors <ins class="diffchange diffchange-inline">for our <a href="https://2010.igem.org/Team:Edinburgh/Bacterial">FORTH framework</a></ins>. The maximum response of the sensor is at a wavelength of 660nm.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The red-light sensor (Cph8) contains three parts:</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The red-light sensor (Cph8) contains three parts:</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>In the absence of red light, phosphorylated OmpR activates EnvZ, which in turn promotes transcription from the <i>ompC</i> promoter and represses transcription from the <i>ompF</i> promoter, thus leading to the expression of LacZ (in this case under the regulation of the aforementioned promoter). This catalyses the formation of a black precipitate from S-gal(3,4-cyclohexenoesculetin-β-D-galactopyranoside).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In the absence of red light, phosphorylated OmpR activates EnvZ, which in turn promotes transcription from the <i>ompC</i> promoter and represses transcription from the <i>ompF</i> promoter, thus leading to the expression of LacZ (in this case under the regulation of the aforementioned promoter). This catalyses the formation of a black precipitate from S-gal(3,4-cyclohexenoesculetin-β-D-galactopyranoside).</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>When exposed to red light, an isomerization in the Cph1 and a structure change in the phycocyanobilin (PCB) part of the photoreceptor inactivates the histidine kinase acitity of EnvZ (Figure 1).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>When exposed to red light, an isomerization in the Cph1 and a structure change in the phycocyanobilin (PCB) part of the photoreceptor inactivates the histidine kinase acitity of EnvZ (<ins class="diffchange diffchange-inline"><a href="https://static.igem.org/mediawiki/2010/8/8c/Ed10-RedSensor.png"></ins>Figure 1<ins class="diffchange diffchange-inline"></a></ins>).</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/8/8c/Ed10-RedSensor.png" width="600px"></p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/8/8c/Ed10-RedSensor.png" width="600px"></p><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as Figure 2.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <ins class="diffchange diffchange-inline"><a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png"></ins>Figure 2<ins class="diffchange diffchange-inline"></a></ins>.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png" width="600px"></p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png" width="600px"></p><br></div></td></tr>
</table>JRWK