http://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&feed=atom&action=historyTeam:Edinburgh/Bacterial/Red light producer - Revision history2024-03-29T15:21:53ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=205464&oldid=prevDonal at 02:25, 28 October 20102010-10-28T02:25:24Z<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/Core_repressilator">the <del class="diffchange diffchange-inline">repressilator</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/Core_repressilator">the <ins class="diffchange diffchange-inline">project</ins></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/BioBricks#Bacterial">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#Bacterial">submitted parts</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/Results#Bacterial">results</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/Results#Bacterial">results</a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href="https://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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<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/Human/Epic">the epic</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/Human/Epic">the epic</a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:Edinburgh/<del class="diffchange diffchange-inline">Results#</del>Human"><del class="diffchange diffchange-inline">results</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/Human<ins class="diffchange diffchange-inline">/FutureApps</ins>"><ins class="diffchange diffchange-inline">future applications</ins></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/Human/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/Human/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_producer&diff=203912&oldid=prevJRWK at 01:19, 28 October 20102010-10-28T01:19:41Z<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;"><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/9/90/Ed10-S284T.jpg" width="500px"></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> The light emissions of our S284T mutated firefly luciferase, BioBricked as <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a>.</p><br><br></center></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;"><p><a href="">Figure 4</a> above shows the distinct red colour of the S284T mutated luciferase (taken at pH 4).</p><br></ins></div></td></tr>
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</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=203123&oldid=prevJRWK at 00:56, 28 October 20102010-10-28T00:56:48Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We submitted a mutant luciferase which produces red light, in order to activate the red light sensor part (which responds optimally to 660nm light).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most <ins class="diffchange diffchange-inline"><b></ins>efficient<ins class="diffchange diffchange-inline"></b> </ins>bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We <ins class="diffchange diffchange-inline"><b></ins>submitted<ins class="diffchange diffchange-inline"></b> </ins>a mutant luciferase which produces red light, in order to activate the red light sensor part (which responds optimally to 660nm 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>Previous works such as <a href="#References">Branchini et al. (2007)</a> and <a href="#References">Moradi et al. (2009)</a> have already identified several luciferase mutants that produce red light. We used site-directed mutagenesis on the wild type to produce three different red light mutants, the emission spectra of which are shown in <a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a> and <a href="https://static.igem.org/mediawiki/2010/8/8e/REDluc356ins.jpg">Figure 2</a>:</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Previous works such as <a href="#References">Branchini et al. (2007)</a> and <a href="#References">Moradi et al. (2009)</a> have already identified several luciferase mutants that produce red light. We used site-directed mutagenesis on the wild type to produce three different red light mutants, the emission spectra of which are shown in <a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a> and <a href="https://static.igem.org/mediawiki/2010/8/8e/REDluc356ins.jpg">Figure 2</a>:</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><p>As stated above, we used site-directed mutagenesis on the <del class="diffchange diffchange-inline">wild type </del>to produce three different red light mutants. We successfully produced two red mutants of the firefly luciferase: mutants 356K and S284T. Both of these glow a nice red colour, but S284T glows much brighter and should be used for work where red coloured bioluminescence is required. Measurements of bioluminescence / OD showed that the S284T luciferase glows at about 35% of the brightness of the wild type green one. The 356K luciferase glows significantly less and is very hard to see, even in a dark room.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>As stated above, we used site-directed mutagenesis on the <ins class="diffchange diffchange-inline">wildtype </ins>to produce three different red light mutants. We <ins class="diffchange diffchange-inline"><b></ins>successfully<ins class="diffchange diffchange-inline"></b> </ins>produced two red mutants of the firefly luciferase: mutants 356K and S284T. Both of these glow a nice red colour, but S284T glows much brighter and should be used for <ins class="diffchange diffchange-inline"><b></ins>work<ins class="diffchange diffchange-inline"></b> </ins>where red coloured bioluminescence is required. Measurements of bioluminescence / OD showed that the S284T luciferase glows at about 35% of the brightness of the wild type green one. The 356K luciferase glows significantly less and is very hard to see, even in a dark room.</p><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="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>This part is one of the only ones with no major setbacks. The main problem will be attempting to activate the red light sensor with something which might not be bright enough. We did not have time to attempt this over the summer. If this does not work, it would be interesting to see if one can combine the codon optimised green luciferase which has been mutated for increased brightness with the mutations for red light.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>This part is one of the only ones with no major <ins class="diffchange diffchange-inline"><b></ins>setbacks<ins class="diffchange diffchange-inline"></b></ins>. The main problem will be attempting to activate the red light sensor with something which might not be bright enough. We did not have time to attempt this over the summer. If this does not work, it would be <ins class="diffchange diffchange-inline"><b></ins>interesting<ins class="diffchange diffchange-inline"></b> </ins>to see if one can combine the codon optimised green luciferase which has been mutated for increased brightness with the mutations for 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: #ffa; color:black; font-size: smaller;"><div><p>Another evident problem is that the luciferin necessary for luciferase activity cannot yet be produced within an <i>E. coli</i> chassis, and thus needs to be added externally. This is mitigated somewhat by the development of BioBricked luciferin-recycling enzymes by <a href="https://2010.igem.org/Team:Cambridge">Cambridge 2010</a>. The genes required for not known, but when they are discovered their addition to this system would improve it greatly.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Another evident <ins class="diffchange diffchange-inline"><b></ins>problem<ins class="diffchange diffchange-inline"></b> </ins>is that the luciferin necessary for luciferase activity cannot yet be produced within an <i>E. coli</i> chassis, and thus needs to be added externally. This is mitigated somewhat by the <ins class="diffchange diffchange-inline"><b></ins>development<ins class="diffchange diffchange-inline"></b> </ins>of BioBricked luciferin-recycling enzymes by <a href="https://2010.igem.org/Team:Cambridge">Cambridge 2010</a>. The genes required for <ins class="diffchange diffchange-inline">it are </ins>not <ins class="diffchange diffchange-inline">currently </ins>known, but when they are discovered their addition to this system would <ins class="diffchange diffchange-inline"><b></ins>improve<ins class="diffchange diffchange-inline"></b> </ins>it greatly.</p></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;"></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>Our BioBricks for this component of the project consist of the two successfully mutated luciferases S284T and 356K, and their composite constructs.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Our BioBricks for this component of the project consist of the two <ins class="diffchange diffchange-inline"><b></ins>successfully<ins class="diffchange diffchange-inline"></b> </ins>mutated luciferases S284T and 356K, and their composite constructs.</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_K322246">BBa_K322246</a>: firefly luciferase from <i>Photinus pyralis</i>, S284T mutant.</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_K322246">BBa_K322246</a>: firefly luciferase from <i>Photinus pyralis</i>, S284T mutant.</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>Figure 3:</b> Results of spectrum analysis of our S284T mutated firefly luciferase, BioBricked as <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a>.</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> Results of spectrum analysis of our S284T mutated firefly luciferase, BioBricked as <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</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><a href="https://static.igem.org/mediawiki/2010/1/18/Ed10-MTLucSpecChar.jpg">Figure 3</a> shows the results of the spectral analysis of the S284T mutated firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a>. The emission spectrum is very close to that shown in the literature (<a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a>), which proves that our mutations have been successful.</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/2010/1/18/Ed10-MTLucSpecChar.jpg">Figure 3</a> shows the results of the spectral analysis of the S284T mutated firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a>. The emission spectrum is very close to that shown in the literature (<a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a>), which proves that our mutations have been <ins class="diffchange diffchange-inline"><b></ins>successful<ins class="diffchange diffchange-inline"></b></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: #ffa; color:black; font-size: smaller;"><div><p>Two major points that need to be emphasised when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity <del class="diffchange diffchange-inline"> </del>of the protein became evident, since the cells were a lot brighter<del class="diffchange diffchange-inline">. Rather </del>than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Two major points that need to be <ins class="diffchange diffchange-inline"><b></ins>emphasised<ins class="diffchange diffchange-inline"></b> </ins>when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter <ins class="diffchange diffchange-inline">- rather </ins>than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</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>pH sensitivity of the <i>Photinus pyralis</i> luciferase has been reported previously <a href="#References">(Seliger and McElroy, 1964)</a>. The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>pH sensitivity of the <i>Photinus pyralis</i> luciferase has been reported previously <a href="#References">(Seliger and McElroy, 1964)</a>. The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><br></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=187968&oldid=prevWillR at 16:36, 27 October 20102010-10-27T16:36:52Z<p></p>
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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>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We <del class="diffchange diffchange-inline">attempted to produce </del>a mutant luciferase which <del class="diffchange diffchange-inline">would produce </del>red light, in order to activate the red light sensor part (which responds optimally to 660nm light).</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We <ins class="diffchange diffchange-inline">submitted </ins>a mutant luciferase which <ins class="diffchange diffchange-inline">produces </ins>red light, in order to activate the red light sensor part (which responds optimally to 660nm 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>Previous works such as <a href="#References">Branchini et al. (2007)</a> and <a href="#References">Moradi et al. (2009)</a> have already identified several luciferase mutants that produce red light. We used site-directed mutagenesis on the wild type to produce three different red light mutants, the emission spectra of which are shown in <a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a> and <a href="https://static.igem.org/mediawiki/2010/8/8e/REDluc356ins.jpg">Figure 2</a>:</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Previous works such as <a href="#References">Branchini et al. (2007)</a> and <a href="#References">Moradi et al. (2009)</a> have already identified several luciferase mutants that produce red light. We used site-directed mutagenesis on the wild type to produce three different red light mutants, the emission spectra of which are shown in <a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a> and <a href="https://static.igem.org/mediawiki/2010/8/8e/REDluc356ins.jpg">Figure 2</a>:</p></div></td></tr>
</table>WillRhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=187649&oldid=prevWillR at 16:24, 27 October 20102010-10-27T16:24:13Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>This part is one of the only ones with no major setbacks. The main problem will be attempting to activate the red light sensor with something which might not be bright enough. We did not have time to attempt this over the summer. If this does not work, it would be interesting to see if one can combine the codon optimised green luciferase which has been mutated for increased brightness with the mutations for red light.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>This part is one of the only ones with no major setbacks. The main problem will be attempting to activate the red light sensor with something which might not be bright enough. We did not have time to attempt this over the summer. If this does not work, it would be interesting to see if one can combine the codon optimised green luciferase which has been mutated for increased brightness with the mutations for 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: #ffa; color:black; font-size: smaller;"><div><p>Another evident problem is that the luciferin necessary for luciferase activity <del class="diffchange diffchange-inline">has never been reproduced </del>within an <i>E. coli</i> chassis, and thus needs to be added externally <del class="diffchange diffchange-inline">to stimulate the production of light</del>. <del class="diffchange diffchange-inline">But this </del>is mitigated somewhat by the development of BioBricked luciferin-recycling enzymes by <a href="https://2010.igem.org/Team:Cambridge">Cambridge 2010</a>, <del class="diffchange diffchange-inline">which could theoretically be used in combination with </del>this <del class="diffchange diffchange-inline">luciferase to ensure self-recycling of the required substrates</del>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Another evident problem is that the luciferin necessary for luciferase activity <ins class="diffchange diffchange-inline">cannot yet be produced </ins>within an <i>E. coli</i> chassis, and thus needs to be added externally. <ins class="diffchange diffchange-inline">This </ins>is mitigated somewhat by the development of BioBricked luciferin-recycling enzymes by <a href="https://2010.igem.org/Team:Cambridge">Cambridge 2010</a><ins class="diffchange diffchange-inline">. The genes required for not known</ins>, <ins class="diffchange diffchange-inline">but when they are discovered their addition to </ins>this <ins class="diffchange diffchange-inline">system would improve it greatly</ins>.</p></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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</table>WillRhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=184188&oldid=prevJRWK at 14:28, 27 October 20102010-10-27T14:28:29Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two major points that need to be emphasised when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two major points that need to be emphasised when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</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>pH sensitivity of the Photinus pyralis luciferase has been reported previously <a href="#References">(Seliger and McElroy, 1964)</a>. The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>pH sensitivity of the <ins class="diffchange diffchange-inline"><i></ins>Photinus pyralis<ins class="diffchange diffchange-inline"></i> </ins>luciferase has been reported previously <a href="#References">(Seliger and McElroy, 1964)</a>. The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><br></div></td></tr>
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</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=184179&oldid=prevJRWK at 14:28, 27 October 20102010-10-27T14:28:12Z<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/2010/1/18/Ed10-MTLucSpecChar.jpg">Figure 3</a> shows the results of the spectral analysis of the S284T mutated firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a>. The emission spectrum is very close to that shown in the literature (<a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a>), which proves that our mutations have been successful.</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/2010/1/18/Ed10-MTLucSpecChar.jpg">Figure 3</a> shows the results of the spectral analysis of the S284T mutated firefly luciferase <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a>. The emission spectrum is very close to that shown in the literature (<a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a>), which proves that our mutations have been successful.</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>Two major points that need to be <del class="diffchange diffchange-inline">known </del>when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Two major points that need to be <ins class="diffchange diffchange-inline">emphasised </ins>when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</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>pH sensitivity of the Photinus pyralis luciferase has been reported previously <a href="#References">(Seliger and McElroy, 1964)</a>. The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>pH sensitivity of the Photinus pyralis luciferase has been reported previously <a href="#References">(Seliger and McElroy, 1964)</a>. The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><br></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=184143&oldid=prevJRWK at 14:26, 27 October 20102010-10-27T14:26:57Z<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><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/1/18/Ed10-MTLucSpecChar.jpg" width="500px"></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/2010/1/18/Ed10-MTLucSpecChar.jpg" width="500px"></p><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><b>Figure 3:</b> Results of spectrum analysis of our S284T mutated firefly luciferase.</p><br><br></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><b>Figure 3:</b> Results of spectrum analysis of our S284T mutated firefly luciferase<ins class="diffchange diffchange-inline">, BioBricked as <a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a></ins>.</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/2010/1/18/Ed10-MTLucSpecChar.jpg">Figure 3</a> shows the results of the spectral analysis of the S284T mutated firefly luciferase.</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/2010/1/18/Ed10-MTLucSpecChar.jpg">Figure 3</a> shows the results of the spectral analysis of the S284T mutated firefly luciferase <ins class="diffchange diffchange-inline"><a href="http://partsregistry.org/Part:BBa_K322246">BBa_K322246</a>. The emission spectrum is very close to that shown in the literature (<a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a>), which proves that our mutations have been successful</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>Two major points that need to be known when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two major points that need to be known when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=184045&oldid=prevJRWK at 14:23, 27 October 20102010-10-27T14:23:29Z<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><p>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We attempted to produce a mutant luciferase which would produce red light, in order to activate the red light sensor part (which responds optimally to 660nm light).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We attempted to produce a mutant luciferase which would produce red light, in order to activate the red light sensor part (which responds optimally to 660nm 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: #ffa; color:black; font-size: smaller;"><div><p>Previous works such as <a href="#References">Branchini et al. (2007)</a> and <a href="#References">Moradi et al. (2009)</a> have already identified several luciferase mutants that produce red light. We used site-directed mutagenesis on the wild type to produce three different red light mutants:</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Previous works such as <a href="#References">Branchini et al. (2007)</a> and <a href="#References">Moradi et al. (2009)</a> have already identified several luciferase mutants that produce red light. We used site-directed mutagenesis on the wild type to produce three different red light mutants<ins class="diffchange diffchange-inline">, the emission spectra of which are shown in <a href="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg">Figure 1</a> and <a href="https://static.igem.org/mediawiki/2010/8/8e/REDluc356ins.jpg">Figure 2</a></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><ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><br><p><img src="https://static.igem.org/mediawiki/2010/1/18/Ed10-MTLucSpecChar.jpg" width="500px"></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/2010/1/18/Ed10-MTLucSpecChar.jpg" width="500px"></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">2</del>:</b> Results of spectrum analysis of our S284T mutated firefly luciferase.</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">3</ins>:</b> Results of spectrum analysis of our S284T mutated firefly luciferase.</p><br><br></center<ins class="diffchange diffchange-inline">></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><a href="https://static.igem.org/mediawiki/2010/1/18/Ed10-MTLucSpecChar.jpg">Figure 3</a> shows the results of the spectral analysis of the S284T mutated firefly luciferase.</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: #eee; color:black; font-size: smaller;"><div><p>Two major points that need to be known when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two major points that need to be known when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td></tr>
</table>JRWKhttp://2010.igem.org/wiki/index.php?title=Team:Edinburgh/Bacterial/Red_light_producer&diff=182332&oldid=prevJRWK at 13:22, 27 October 20102010-10-27T13:22:16Z<p></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">Revision as of 13:22, 27 October 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We attempted to produce a mutant luciferase which would produce red light, in order to activate the red light sensor part (which responds optimally to 660nm light).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Firefly luciferase (EC <a href="http://www.expasy.org/cgi-bin/nicezyme.pl?1.13.12.7">1.13.12.7</a>) from <a href="http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&amp;search_value=722476" ><i>Photinus pyralis</i></a> is one of the most efficient bioluminescent proteins known. Its emission peak is about 557nm at pH 7.8 (this is the ordinary internal pH of <i>E. coli</i> during growth). We attempted to produce a mutant luciferase which would produce red light, in order to activate the red light sensor part (which responds optimally to 660nm 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: #ffa; color:black; font-size: smaller;"><div><p>We used site-directed mutagenesis on the wild type to produce three different red light mutants:</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><ins class="diffchange diffchange-inline">Previous works such as <a href="#References">Branchini et al. (2007)</a> and <a href="#References">Moradi et al. (2009)</a> have already identified several luciferase mutants that produce red light. </ins>We used site-directed mutagenesis on the wild type to produce three different red light mutants:</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><ul></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><center><br><p><img src="https://static.igem.org/mediawiki/2010/d/d9/REDlucS248Tspectrum.jpg" border="0" /></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/d/d9/REDlucS248Tspectrum.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 1:</b> Emission spectra of the <i>P. pyralis</i> luciferase mutant S284T.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 1:</b> Emission spectra of the <i>P. pyralis</i> luciferase mutant S284T.</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Image: Branchini et al. (2007)</p><br><br></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Image: <ins class="diffchange diffchange-inline"><a href="#References"></ins>Branchini et al. (2007)<ins class="diffchange diffchange-inline"></a></ins></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: #eee; color:black; font-size: smaller;"><div><center><br><p><img src="https://static.igem.org/mediawiki/2010/8/8e/REDluc356ins.jpg" width="600" border="0" /></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/2010/8/8e/REDluc356ins.jpg" width="600" 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 2:</b> Emission spectra of the <i>P. pyralis</i> luciferase mutants 356R (1) and 356K (2).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 2:</b> Emission spectra of the <i>P. pyralis</i> luciferase mutants 356R (1) and 356K (2).</p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Image: Moradi et al. (2009)</p><br><br></center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Image: <ins class="diffchange diffchange-inline"><a href="#References"></ins>Moradi et al. (2009)<ins class="diffchange diffchange-inline"></a></ins></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: #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>Two major points that need to be known when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Two major points that need to be known when using this BioBrick are the temperature sensitivity of the luciferase, and its pH sensitivity. During the first part of the project, the cells were grown at 37C. When we tested growing them at 30C, the temperature sensitivity of the protein became evident, since the cells were a lot brighter. Rather than waiting 10 minutes in the dark room to get our eyes accustomed, they were visible before the door was closed.</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>pH sensitivity of the Photinus pyralis luciferase has been reported previously (Seliger and McElroy, 1964). The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>pH sensitivity of the Photinus pyralis luciferase has been reported previously <ins class="diffchange diffchange-inline"><a href="#References"></ins>(Seliger and McElroy, 1964)<ins class="diffchange diffchange-inline"></a></ins>. The cells were usually suspended in citrate buffer, pH 4.8, as this allows the luciferin to enter the cells faster. This has an effect on the colour emitted, though not as marked as for the wildtype.</p><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>
</table>JRWK