Team:Edinburgh/Bacterial/Blue light producer

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   <li><a href="http://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Project/Future">the future</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Project/References">references</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Project/References">references</a></li>
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a>
  <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the project</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Future">the future</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Modelling/Future">the future</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li>
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a>
  <li><a href="http://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a>
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li>
 
   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Terminology">terminology research</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Branding">iGEM survey</a></li>
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Wiki">wiki</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li>
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Identity">identity</a></li>
 
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Collaboration">collaboration</a></li>
 
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Branding">branding research</a></li>
 
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/SciFi">science fiction writing</a></li>
 
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li>
 
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  <li><a href="http://2010.igem.org/Team:Edinburgh/Human/SelfReflection">self-reflection</a></li>
 
   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li>
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   <li><a href="http://2010.igem.org/Team:Edinburgh/Results#Human">further thoughts</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/References">references</a></li>
   <li><a href="http://2010.igem.org/Team:Edinburgh/Human/References">references</a></li>
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<p>The construction of a blue light producer requires the combination of LuxAB with an extra protein called lumazine (LumP). According to O'Kane and Lee (1985), this will shift the wavelength of the bacterial luciferase towards the blue spectrum (from 495 nm to 478 nm), giving us a light which can hopefully activate our <a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue light sensor</a>. <a href="http://2009.igem.org/Team:Edinburgh">Last year's Edinburgh team</a> already made both of these parts individually, so all we had to do was fuse them together and add a constitutive promoter before determining whether the wavelength of the light output had been altered.</p>
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<p>To produce blue light, we used two systems using the bacterial luciferase from <i>Xenorhabdus luminescens</i> (LuxAB). The standalone LuxAB has an emission peak of 495nm (<a href="http://2010.igem.org/wiki/images/0/09/Ed10-LuxABSpectra.gif">Figure 1</a>). According to <a href="#References">O'Kane and Lee (1985)</a> and others, the addition of lumazine protein LumP shifts the wavelength towards the blue spectrum (478 nm), such that it will be in the correct region to <b>activate</b> the <a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue light sensor</a>. </p>
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<center><br><p><img src="http://2010.igem.org/wiki/images/0/09/Ed10-LuxABSpectra.gif" border="0"/></p>
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<center><br><br><p><img src="http://2010.igem.org/wiki/images/0/09/Ed10-LuxABSpectra.gif" border="0"/></p><br>
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<p><b>Figure 1:</b> Emission spectra of LuxAB (in solid black line). The LuxAB depicted is from <i>V. campbellii</i>, but sources confirm that other bacterial luciferases produce similar graphs.</p>
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<p><b>Figure 1:</b> Emission spectra of LuxAB (in solid black line). The LuxAB depicted is that from <i>Vibrio campbellii</i>, but sources confirm that other bacterial luciferases produce similar spectral graphs.</p>
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<p>Image: Suadee et al. (2008)</p><br><br></center>
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<p>Image: <a href="#References">Suadee et al. (2008)</a></p><br><br></center>
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<a name="Strategy" id="Strategy"></a><h2>Strategy</h2>
<a name="Strategy" id="Strategy"></a><h2>Strategy</h2>
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<p>***</p>
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<p><a href="http://2009.igem.org/Team:Edinburgh">Last year's Edinburgh team</a> already made both of these parts individually, so all we had to do was <b>fuse</b> them together and add a promoter. Individual parts were resubmitted in pSB1C3.</p>
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<p>The LuxAB which last year's team made and stored is failing to transform correctly. Colonies that ought to be growing white are growing blue (LuxAB should have replaced the <i>lacZ'</i> in the vector, EdinbrickI). A similar thing is happening with LumP and RFP, where cells are still growing red, despite the fact that the RFP ought to have been removed when LumP was inserted last year.</p>
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<p>We were <b>experiencing</b> problems with last year’s <a href="http://partsregistry.org/Part:BBa_K216008">LuxAB</a> and <a href="http://partsregistry.org/Part:BBa_K216007">LumP</a> parts, in that blue and red colonies were growing when they should not be. This was another reason for <b>resubmitting</b> them, as the parts contained in the Registry may have the same problems.</p>
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<p>We have come across several PCR products not yet inserted into vectors with which we are hoping to start this part of the project (i.e. the bit which should already have been done by last year according to their lab notes) again.</p>
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<p>We eventually resolved these problems by using the alternative PCR products which were perfectly capable of being cloned into pSB1C3.</p>
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<p>We have also had issues with the instrument used to <b>measure</b> the spectral output. It could not pick up a signal from the tubes despite visible bioluminescence. Hopefully we will be able to characterise these parts more fully before the Jamboree.</p>
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<p>Both <i>lumP</i> and <i>luxAB</i> have already been submitted as BioBricks in pSB1A3 by <a href="http://2009.igem.org/Team:Edinburgh">last year's Edinburgh team</a>, but we are re-submitting slightly modified versions in pSB1C3.</p>
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<p>Both <i>lumP</i> and <i>luxAB</i> have already been submitted as BioBricks in pSB1A3 by <a href="http://2009.igem.org/Team:Edinburgh">last year's Edinburgh team</a>, but we are re-submitting <b>corrected</b> variants in pSB1C3.</p>
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<p>We are also submitting a luxAB-lumP fusion construct in pSB1C3 with a constitutive promoter which can be used for blue light production. We have confirmation of working luxAB with the promoter in question.</p>
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<p>We are also submitting a luxAB-lumP fusion construct in pSB1C3 with a constitutive promoter which can be used for blue light production. We have confirmation of working luxAB with the promoter in question.</p><br>
<p><a href="http://partsregistry.org/Part:BBa_K322139">BBa_K322139</a>: bacterial luciferase <i>luxAB</i>, updated version of <a href="http://partsregistry.org/Part:BBa_K216008">BBa_K216008</a>.</p>
<p><a href="http://partsregistry.org/Part:BBa_K322139">BBa_K322139</a>: bacterial luciferase <i>luxAB</i>, updated version of <a href="http://partsregistry.org/Part:BBa_K216008">BBa_K216008</a>.</p>
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<p><a href="http://partsregistry.org/Part:BBa_K322149">BBa_K322149</a>: <i>luxAB</i> and <i>lumP</i> composite part.</p>
<p><a href="http://partsregistry.org/Part:BBa_K322149">BBa_K322149</a>: <i>luxAB</i> and <i>lumP</i> composite part.</p>
<p><a href="http://partsregistry.org/Part:BBa_K322150">BBa_K322150</a>: <i>luxAB</i> and <i>lumP</i> under <i>lac</i> promoter.</p><br>
<p><a href="http://partsregistry.org/Part:BBa_K322150">BBa_K322150</a>: <i>luxAB</i> and <i>lumP</i> under <i>lac</i> promoter.</p><br>
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<p>***</p>
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<center><br><br><p><img src="http://2010.igem.org/wiki/images/a/a4/Ed10-blue01.jpg" border="0" width="600px"/></p><br>
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<p><b>Figure 2:</b> Emission output (blue light) of the <i>luxAB/lumP</i> fusion.</p><br><br></center>
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<p><a href="http://2010.igem.org/wiki/images/a/a4/Ed10-blue01.jpg">Figure 2</a> shows the light output of the <i>luxAB/lumP</i> fusion, a distinctly blue colour. Unfortunately, we have been unable so far to get a spectrum reading from our samples, but hope to be able to achieve one by the Jamboree.</p>
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Latest revision as of 02:29, 28 October 2010







Overview: The blue light producer


To produce blue light, we used two systems using the bacterial luciferase from Xenorhabdus luminescens (LuxAB). The standalone LuxAB has an emission peak of 495nm (Figure 1). According to O'Kane and Lee (1985) and others, the addition of lumazine protein LumP shifts the wavelength towards the blue spectrum (478 nm), such that it will be in the correct region to activate the blue light sensor.




Figure 1: Emission spectra of LuxAB (in solid black line). The LuxAB depicted is that from Vibrio campbellii, but sources confirm that other bacterial luciferases produce similar spectral graphs.

Image: Suadee et al. (2008)



Strategy


Last year's Edinburgh team already made both of these parts individually, so all we had to do was fuse them together and add a promoter. Individual parts were resubmitted in pSB1C3.



Problems


We were experiencing problems with last year’s LuxAB and LumP parts, in that blue and red colonies were growing when they should not be. This was another reason for resubmitting them, as the parts contained in the Registry may have the same problems.

We have also had issues with the instrument used to measure the spectral output. It could not pick up a signal from the tubes despite visible bioluminescence. Hopefully we will be able to characterise these parts more fully before the Jamboree.



BioBricks


Both lumP and luxAB have already been submitted as BioBricks in pSB1A3 by last year's Edinburgh team, but we are re-submitting corrected variants in pSB1C3.

We are also submitting a luxAB-lumP fusion construct in pSB1C3 with a constitutive promoter which can be used for blue light production. We have confirmation of working luxAB with the promoter in question.


BBa_K322139: bacterial luciferase luxAB, updated version of BBa_K216008.

BBa_K322312: luxCDE (required for luxAB expression), updated version of BBa_K216017.

BBa_K322007: lumP (shifts luxAB to blue), updated version of BBa_K216007.

BBa_K322140: luxAB under lac promoter.

BBa_K322141: luxAB and luxCDE under lac promoter.

BBa_K322149: luxAB and lumP composite part.

BBa_K322150: luxAB and lumP under lac promoter.





Characterisation





Figure 2: Emission output (blue light) of the luxAB/lumP fusion.



Figure 2 shows the light output of the luxAB/lumP fusion, a distinctly blue colour. Unfortunately, we have been unable so far to get a spectrum reading from our samples, but hope to be able to achieve one by the Jamboree.



References


O'Kane, D. J. & Lee, J. (1985). Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum. Biochemistry 24, 1467-1475.

Suadee, C., Nijvipakul, S., et al. (2008). LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence. J. Bacteriol. 190(5): 1531-1538

Edinburgh 2009 team wiki, http://2009.igem.org/Team:Edinburgh.




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