http://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&feed=atom&action=historyTeam:Edinburgh/Bacterial/Green light producer - Revision history2024-03-28T12:07:57ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=205633&oldid=prevDonal at 02:34, 28 October 20102010-10-28T02:34:15Z<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/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: #ffa; color:black; font-size: smaller;"><div><p>Firefly luciferase was originally deposited in the Registry by <a href="<del class="diffchange diffchange-inline">http</del>://<del class="diffchange diffchange-inline">parts</del>.<del class="diffchange diffchange-inline">mit</del>.<del class="diffchange diffchange-inline">edu/igem07/index.php</del>/Ljubljana">Ljubljana 2007</a>. We modified this slightly and re-submitted in pSB1C3, along with a codon-optimised mutant and a simple reporter system.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Firefly luciferase was originally deposited in the Registry by <a href="<ins class="diffchange diffchange-inline">https</ins>://<ins class="diffchange diffchange-inline">2007</ins>.<ins class="diffchange diffchange-inline">igem</ins>.<ins class="diffchange diffchange-inline">org</ins>/Ljubljana">Ljubljana 2007</a>. We modified this slightly and re-submitted in pSB1C3, along with a codon-optimised mutant and a simple 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><a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>: firefly luciferase from <i>Photinus pyralis</i>, modified <a href="http://partsregistry.org/Part:BBa_I712019">BBa_I712019</a>.</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_K322237">BBa_K322237</a>: firefly luciferase from <i>Photinus pyralis</i>, modified <a href="http://partsregistry.org/Part:BBa_I712019">BBa_I712019</a>.</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><b>Fujii, H., Noda, K., Asami, Y., Kuroda, A., Sakata, M. & Tokida, A. (2007).</b> Increase in bioluminescence intensity of firefly luciferase using genetic modification. <i>Analytical Biochemistry</i> <b>366</b>, 131-136.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Fujii, H., Noda, K., Asami, Y., Kuroda, A., Sakata, M. & Tokida, A. (2007).</b> Increase in bioluminescence intensity of firefly luciferase using genetic modification. <i>Analytical Biochemistry</i> <b>366</b>, 131-136.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Shapirol, E., Lu, C., Baneyx, F. (2009).</b> Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis. <i>Protein Eng Des Sel</i> <b>22</b>(11): 655-663.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Shapirol, E., Lu, C., Baneyx, F. (2009).</b> Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis. <i>Protein Eng Des Sel</i> <b>22</b>(11): 655-663.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Ljubljana 2007 team wiki, <i><del class="diffchange diffchange-inline">http</del>://<del class="diffchange diffchange-inline">parts</del>.<del class="diffchange diffchange-inline">mit</del>.<del class="diffchange diffchange-inline">edu/igem07/index.php</del>/Ljubljana</i>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Ljubljana 2007 team wiki, <i><ins class="diffchange diffchange-inline">https</ins>://<ins class="diffchange diffchange-inline">2007</ins>.<ins class="diffchange diffchange-inline">igem</ins>.<ins class="diffchange diffchange-inline">org</ins>/Ljubljana</i>.</p></div></td></tr>
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</table>Donalhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=203590&oldid=prevJRWK at 01:11, 28 October 20102010-10-28T01:11:02Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>For the production of green light, we relied on tried and trusted firefly luciferase from <i>P. pyralis</i>. This has a recorded luminescence emission peak of roughly 557nm, as recorded in <a href="#References">Shapiro et al. (2009)</a> and other references (<a href="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg">Figure 1</a>). This part was originally submitted by Ljubljana 2007. The sequence analysis revealed that there were 6 bases inserted upstream of the coding sequence. The distance between the ribosome binding site and the start codon was thus not optimal, and this resulted in a decreased luminescence intensity of the cells. We removed these 6 bases by PCR and resubmitted the part in pSB1C3.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>For the <ins class="diffchange diffchange-inline"><b></ins>production<ins class="diffchange diffchange-inline"></b> </ins>of green light, we relied on tried and trusted firefly luciferase from <i>P. pyralis</i>. This has a <ins class="diffchange diffchange-inline"><b></ins>recorded<ins class="diffchange diffchange-inline"></b> </ins>luminescence emission peak of roughly 557nm, as recorded in <a href="#References">Shapiro et al. (2009)</a> and other <ins class="diffchange diffchange-inline"><b></ins>references<ins class="diffchange diffchange-inline"></b> </ins>(<a href="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg">Figure 1</a>). This part was originally <ins class="diffchange diffchange-inline"><b></ins>submitted<ins class="diffchange diffchange-inline"></b> </ins>by Ljubljana 2007. The sequence <ins class="diffchange diffchange-inline"><b></ins>analysis<ins class="diffchange diffchange-inline"></b> </ins>revealed that there were 6 bases inserted upstream of the coding sequence. The distance between the ribosome binding site and the start codon was thus not <ins class="diffchange diffchange-inline"><b></ins>optimal<ins class="diffchange diffchange-inline"></b></ins>, and this resulted in a decreased luminescence intensity of the cells. We removed these 6 bases by PCR and <ins class="diffchange diffchange-inline"><b></ins>resubmitted<ins class="diffchange diffchange-inline"></b> </ins>the part in pSB1C3.</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><center><br><p><img src="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg" border="0" width="500px"/></p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><p><img src="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg" border="0" width="500px"/></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>Image: <a href="#References">Shapiro et al. (2009)</a></p><br><br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Image: <a href="#References">Shapiro et al. (2009)</a></p><br><br></center></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>An alternative approach was planned during the early stages of the project: the combination of <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">LuxAB</a> with another protein: YFP from <i>Vibrio fischeri</i>. This would <b>shift</b> the wavelength produced by LuxAB towards the green regions of the spectrum, to <b>activate</b> our green light sensor. During our discussions with <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">UNAM-Genomics Mexico</a> we realised that they planned on making the same part, so we stopped working on it, planning on testing its <b>combination</b> with our green light sensor later on. We unfortunately have not yet reached that stage, but the part is available in the registry as <a href="http://partsregistry.org/Part:BBa_K360010">BBa_K360010</a>.</p></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 style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </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;"><p>An alternative approach was planned during the early stages of the project: The combination of <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">LuxAB</a> with another protein: YFP from <i>Vibrio fischeri</i>. This will shift the wavelength produced by LuxAB towards the green regions of the spectrum, to activate our green light sensor. During our discussions with <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">UNAM-Genomics Mexico</a> we realised that they planned on making the same part, so we stopped working on it, planning on testing its combination with our green light sensor later on. We unfortunately did not have time to get to that stage. It is available in the registry as BBa_K360010. </p></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </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;"></del></div></td><td colspan="2"> </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;"><a name="Brighter luciferase" id="Brighter luciferase"></a><h2>LUCIE: A brighter luciferase</h2></del></div></td><td colspan="2"> </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;"><br></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</p></div></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;"><br></del></div></td><td colspan="2"> </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;"><p></del></div></td><td colspan="2"> </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;">LUCIE (LUCiferase with Intense Emission) has three mutations compared to the wild type: Ile423Leu, Asp436Gly, and Leu530Arg. This causes more efficient binding of ATP and luciferin. These are used up faster, resulting in increased brightness of the cells.</p></del></div></td><td colspan="2"> </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;"></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><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p><a href="http://partsregistry.org/Part:BBa_K322451">LUCIE</a> (LUCiferase with Intense Emission) has three mutations compared to the wild type: Ile423Leu, Asp436Gly, and Leu530Arg. This causes more efficient binding of ATP and luciferin, which are used up faster and result in increased brightness of the cells.</p></ins><br></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>
<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>The <del class="diffchange diffchange-inline">wild type </del>version of the luciferase was cloned into pSB1C3. The 6 extra bases between the start codon and the start codon were removed by PCR, using a forward primer starting at the start codon, and an RBS based reverse primer. The part and the vector were thus amplified, but without the 6 extra bases.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The <ins class="diffchange diffchange-inline">wildtype </ins>version of the luciferase was cloned into pSB1C3. The 6 extra bases between the start codon and the start codon were removed by PCR, using a forward primer starting at the start codon, and an RBS based reverse primer. The part and the vector were thus amplified, but without the 6 extra bases.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><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><p>The codon optimised LUCIE luciferase was synthesised by <del class="diffchange diffchange-inline">Geneart</del>, then transferred to pSB1C3.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The codon optimised LUCIE luciferase was <ins class="diffchange diffchange-inline"><b></ins>synthesised<ins class="diffchange diffchange-inline"></b> </ins>by <ins class="diffchange diffchange-inline"><a href="http://www.geneart.com/">GENEART</a></ins>, then transferred to pSB1C3.</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;"><div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><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>
<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><del class="diffchange diffchange-inline">Geneart </del>is overloaded with requests at this time of year. The bright luciferase arrived at the last moment, and the photos were taken the same day as the wiki freeze. We did not have time to characterise it <del class="diffchange diffchange-inline">more</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">Unfortunately, GENEART </ins>is <ins class="diffchange diffchange-inline">often </ins>overloaded with <ins class="diffchange diffchange-inline">synthesis </ins>requests at this time of year. The bright luciferase arrived at the last moment, and the photos were taken the same day as the wiki freeze. We did not have time to characterise it <ins class="diffchange diffchange-inline">further</ins>.</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><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><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><b>Figure 2:</b> Results of spectrum analysis of our wildtype firefly luciferase.</p><br><br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 2:</b> Results of spectrum analysis of our wildtype firefly luciferase.</p><br><br></center></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><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. This is due to the fact that the cells were suspended in citrate buffer, pH4.8, to help the luciferin enter the cells. At pH 7, the luciferase peaks in the yellow green region and does not make red light. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. This is due to the fact that the cells were suspended in citrate buffer, pH4.8, to help the luciferin enter the cells. At pH 7, the luciferase peaks in the yellow green region and does not make red light.</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><a href="https://static.igem.org/mediawiki/2010/5/53/Ed10-green.jpg">Figure 3</a> and <a href="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg">Figure 4</a> on the next page demonstrate the difference in spectral output due to the pH of the buffer solution.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/5/53/Ed10-green.jpg">Figure 3</a> and <a href="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg">Figure 4</a> on the next page demonstrate the difference in spectral output due to the pH of the buffer solution.</p></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=201861&oldid=prevWillR at 00:08, 28 October 20102010-10-28T00:08:34Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 00:08, 28 October 2010</td>
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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>
<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><del class="diffchange diffchange-inline">***</del></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">Geneart is overloaded with requests at this time of year. The bright luciferase arrived at the last moment, and the photos were taken the same day as the wiki freeze. We did not have time to characterise it more.</ins></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><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><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><b>Figure 2:</b> Results of spectrum analysis of our wildtype firefly luciferase.</p><br><br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 2:</b> Results of spectrum analysis of our wildtype firefly luciferase.</p><br><br></center></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><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. <del class="diffchange diffchange-inline">Given that the difference between the aforementioned mutant and this wildtype </del>is <del class="diffchange diffchange-inline">very noticeable </del>to the <del class="diffchange diffchange-inline">naked eye</del>, <del class="diffchange diffchange-inline">it is difficult </del>to <del class="diffchange diffchange-inline">pinpoint </del>the <del class="diffchange diffchange-inline">cause of this discrepancy; we hope to be able to re-run </del>the <del class="diffchange diffchange-inline">analysis </del>in <del class="diffchange diffchange-inline">an attempt to </del>make <del class="diffchange diffchange-inline">sense of it</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. <ins class="diffchange diffchange-inline">This </ins>is <ins class="diffchange diffchange-inline">due </ins>to the <ins class="diffchange diffchange-inline">fact that the cells were suspended in citrate buffer, pH4.8</ins>, to <ins class="diffchange diffchange-inline">help </ins>the <ins class="diffchange diffchange-inline">luciferin enter </ins>the <ins class="diffchange diffchange-inline">cells. At pH 7, the luciferase peaks </ins>in <ins class="diffchange diffchange-inline">the yellow green region and does not </ins>make <ins class="diffchange diffchange-inline">red light</ins>. </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><a href="https://static.igem.org/mediawiki/2010/5/53/Ed10-green.jpg">Figure 3</a> and <a href="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg">Figure 4</a> on the next page demonstrate the difference in spectral output due to the pH of the buffer solution.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/5/53/Ed10-green.jpg">Figure 3</a> and <a href="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg">Figure 4</a> on the next page demonstrate the difference in spectral output due to the pH of the buffer solution.</p></div></td></tr>
</table>WillRhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=199882&oldid=prevJRWK at 23:04, 27 October 20102010-10-27T23:04:50Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. Given that the difference between the aforementioned mutant and this wildtype is very noticeable to the naked eye, it is difficult to pinpoint the cause of this discrepancy; we hope to be able to re-run the analysis in an attempt to make sense of it.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. Given that the difference between the aforementioned mutant and this wildtype is very noticeable to the naked eye, it is difficult to pinpoint the cause of this discrepancy; we hope to be able to re-run the analysis in an attempt to make sense of it.</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;"><p><a href="https://static.igem.org/mediawiki/2010/5/53/Ed10-green.jpg">Figure 3</a> and <a href="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg">Figure 4</a> on the next page demonstrate the difference in spectral output due to the pH of the buffer solution.</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><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg" border="0"/></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/e/ec/Ed10-yellow.jpg" border="0"/></p><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 4:</b> Emission output (yellow light) of the wildtype firefly luciferase in citrate buffer (pH 4.8).</p><br><br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 4:</b> Emission output (yellow light) of the wildtype firefly luciferase in citrate buffer (pH 4.8).</p><br><br></center></div></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;"></del></div></td><td colspan="2"> </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;"><p><a href="https://static.igem.org/mediawiki/2010/5/53/Ed10-green.jpg">Figure 3</a> and <a href="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg">Figure 4</a> demonstrate the difference in spectral output due to the pH of the buffer solution.</p></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><a name="References" id="References"></a><h2>References</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="References" id="References"></a><h2>References</h2></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=199802&oldid=prevJRWK at 23:02, 27 October 20102010-10-27T23:02:42Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. Given that the difference between the aforementioned mutant and this wildtype is very noticeable to the naked eye, it is difficult to pinpoint the cause of this discrepancy; we hope to be able to re-run the analysis in an attempt to make sense of it.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><a href="https://static.igem.org/mediawiki/2010/9/9b/Ed10-WTLucSpecChar.jpg">Figure 2</a> shows the results of the spectral analysis of our wildtype firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322237">BBa_K322237</a>. Unlike the emission spectrum for our red-light producing <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">S248T variant</a>, the spectrum differs greatly from the expected; there is a small peak at roughly the correct location (557nm), but also a shoulder at approximately 580nm and a further peak at 600nm. Given that the difference between the aforementioned mutant and this wildtype is very noticeable to the naked eye, it is difficult to pinpoint the cause of this discrepancy; we hope to be able to re-run the analysis in an attempt to make sense of it.</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>
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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;"><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;"></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/2010/5/53/Ed10-green.jpg" border="0"/></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> Emission output (green light) of the wildtype firefly luciferase in neutral buffer.</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;"><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg" border="0"/></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 4:</b> Emission output (yellow light) of the wildtype firefly luciferase in citrate buffer (pH 4.8).</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/2010/5/53/Ed10-green.jpg">Figure 3</a> and <a href="https://static.igem.org/mediawiki/2010/e/ec/Ed10-yellow.jpg">Figure 4</a> demonstrate the difference in spectral output due to the pH of the buffer solution.</p></ins></div></td></tr>
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</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=195033&oldid=prevWillR at 20:23, 27 October 20102010-10-27T20:23:36Z<p></p>
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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><del class="diffchange diffchange-inline">***</del></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">The wild type version of the luciferase was cloned into pSB1C3. The 6 extra bases between the start codon and the start codon were removed by PCR, using a forward primer starting at the start codon, and an RBS based reverse primer. The part and the vector were thus amplified, but without the 6 extra bases.</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"><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>The codon optimised LUCIE luciferase was synthesised by Geneart, then transferred to pSB1C3.</ins></div></td></tr>
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</table>WillRhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=193839&oldid=prevWillR at 19:40, 27 October 20102010-10-27T19:40:45Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</p></div></td></tr>
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</table>WillRhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=193296&oldid=prevWillR at 19:22, 27 October 20102010-10-27T19:22:09Z<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;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><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;">LUCIE (LUCiferase with Intense Emission) has three mutations compared to the wild type: Ile423Leu, Asp436Gly, and Leu530Arg. This causes more efficient binding of ATP and luciferin. These are used up faster, resulting in increased brightness of the cells.</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;"></ins></div></td></tr>
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</table>WillRhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=193027&oldid=prevWillR at 19:13, 27 October 20102010-10-27T19:13:30Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Image: <a href="#References">Shapiro et al. (2009)</a></p><br><br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Image: <a href="#References">Shapiro et al. (2009)</a></p><br><br></center></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;"></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>An alternative approach was planned during the early stages of the project: The combination of <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">LuxAB</a> with another protein: YFP from <i>Vibrio fischeri</i>. This will shift the wavelength produced by LuxAB towards the green regions of the spectrum, to activate our green light sensor. During our discussions with <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">UNAM-Genomics Mexico</a> we realised that they planned on making the same part, so we stopped working on it, planning on testing its combination with our green light sensor later on. We unfortunately did not have time to get to that stage. It is available in the registry as BBa_K360010. </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;"></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;"><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;"><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;"></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;"><a name="Brighter luciferase" id="Brighter luciferase"></a><h2>LUCIE: A brighter luciferase</h2></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;"><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</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;"><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;"><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">p>An alternative approach involved the combination of <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">LuxAB</a> with the production of another protein: YFP from <i>Vibrio fischeri</i>. This will shift the wavelength of the blue light produced by LuxAB towards the yellow spectrum, making it the correct colour to activate a green light sensor. We expect our main collaborators, <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">UNAM-Genomics Mexico</a> to synthesise this DNA sequence and combine it with LuxAB. We also expect UNAM-Genomics to BioBrick it as part of their submission to the Registry.</</del>p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p></div></td></tr>
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</table>WillRhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Green_light_producer&diff=190872&oldid=prevWillR at 18:07, 27 October 20102010-10-27T18:07:18Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 18:07, 27 October 2010</td>
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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><p>For the production of green light, we relied on tried and trusted firefly luciferase from <i>P. pyralis</i>. This has a recorded luminescence emission peak of roughly 557nm, as recorded in <a href="#References">Shapiro et al. (2009)</a> and other references (<a href="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg">Figure 1</a>).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>For the production of green light, we relied on tried and trusted firefly luciferase from <i>P. pyralis</i>. This has a recorded luminescence emission peak of roughly 557nm, as recorded in <a href="#References">Shapiro et al. (2009)</a> and other references (<a href="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg">Figure 1</a>)<ins class="diffchange diffchange-inline">. This part was originally submitted by Ljubljana 2007. The sequence analysis revealed that there were 6 bases inserted upstream of the coding sequence. The distance between the ribosome binding site and the start codon was thus not optimal, and this resulted in a decreased luminescence intensity of the cells. We removed these 6 bases by PCR and resubmitted the part in pSB1C3</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><p><img src="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg" border="0" width="500px"/></p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><p><img src="https://static.igem.org/mediawiki/2010/6/6a/Ed10-FireflyLucSpectra.jpg" border="0" width="500px"/></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>Image: <a href="#References">Shapiro et al. (2009)</a></p><br><br></center></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Image: <a href="#References">Shapiro et al. (2009)</a></p><br><br></center></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>In addition to the above, we created <del class="diffchange diffchange-inline">and characterised </del>a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be <del class="diffchange diffchange-inline">up to </del>12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>In addition to the above, we created a codon-optimised mutant of the firefly luciferase, reported by <a href="#References">Fujii et al. (2007)</a> to be 12.5 times brighter than the wildtype. This may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.</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>An alternative approach involved the combination of <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">LuxAB</a> with the production of another protein: YFP from <i>Vibrio fischeri</i>. This will shift the wavelength of the blue light produced by LuxAB towards the yellow spectrum, making it the correct colour to activate a green light sensor. We expect our main collaborators, <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">UNAM-Genomics Mexico</a> to synthesise this DNA sequence and combine it with LuxAB. We also expect UNAM-Genomics to BioBrick it as part of their submission to the Registry.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>An alternative approach involved the combination of <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">LuxAB</a> with the production of another protein: YFP from <i>Vibrio fischeri</i>. This will shift the wavelength of the blue light produced by LuxAB towards the yellow spectrum, making it the correct colour to activate a green light sensor. We expect our main collaborators, <a href="https://2010.igem.org/Team:UNAM-Genomics_Mexico">UNAM-Genomics Mexico</a> to synthesise this DNA sequence and combine it with LuxAB. We also expect UNAM-Genomics to BioBrick it as part of their submission to the Registry.</p></div></td></tr>
</table>WillR