Team:Edinburgh/Bacterial/Green light producer

From 2010.igem.org

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<p>The green luciferase will require the combination of luxAB with the production of another protein: YFP from ''Vibrio fischeri''. This will shift the wavelength of the light towards the yellow spectrum, making it the correct colour to activate our green light sensor. Mexico UNAM-Genomics have had this DNA sequence synthesised and sending it to us for characterisation and combination with luxAB. We also expect Mexico to BioBrick it as part of their submission to the registry.</p>
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<p>For the production of green light, we will rely on tried and trusted firefly luciferase from <i>P. pyralis</i>. This has a recorded luminescence emission peak of roughly 550 nm, as recorded in Shapiro et al. (2009) and other references.</p>
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<center><br><p><img src="http://2010.igem.org/wiki/images/6/6a/Ed10-FireflyLucSpectra.jpg" border="0" width="500px"/></p>
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<p><b>Figure 1:</b> Emission spectra of the <i>P. pyralis</i> luciferase (in solid black line).</p>
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<p>Image: Shapiro et al. (2009)</p><br><br></center>
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<p>In addition to the above, we will attempt to create and characterise a codon-optimised mutant of the firefly luciferase, as reported by Fujii et al. (2007) to be up to 12.5 times brighter than the wildtype. If successful, 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>
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<p>An alternative approach involves the combination of <a href="http://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="">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>
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<a name="Problems" id="Problems"></a><h2>Problems</h2>
<a name="Problems" id="Problems"></a><h2>Problems</h2>
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<p>You can't have problems with a piece of DNA you've never met.</p>
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<p>***</p>
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<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>
<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>
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<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>
<p>Ljubljana 2007 team wiki, <i>http://parts.mit.edu/igem07/index.php/Ljubljana</i>.</p>
<p>Ljubljana 2007 team wiki, <i>http://parts.mit.edu/igem07/index.php/Ljubljana</i>.</p>

Revision as of 13:46, 26 October 2010







Overview: The green light producer


For the production of green light, we will rely on tried and trusted firefly luciferase from P. pyralis. This has a recorded luminescence emission peak of roughly 550 nm, as recorded in Shapiro et al. (2009) and other references.


Figure 1: Emission spectra of the P. pyralis luciferase (in solid black line).

Image: Shapiro et al. (2009)



In addition to the above, we will attempt to create and characterise a codon-optimised mutant of the firefly luciferase, as reported by Fujii et al. (2007) to be up to 12.5 times brighter than the wildtype. If successful, this may help to alleviate the problems foreseen with a lack of luminescence intensity failing to activate the light sensors of the corresponding wavelength.

An alternative approach involves the combination of LuxAB with the production of another protein: YFP from Vibrio fischeri. 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, UNAM-Genomics Mexico 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.



Strategy


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Problems


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BioBricks


Firefly luciferase was originally deposited in the Registry by Ljubljana 2007. We modified this slightly and re-submitted in pSB1C3, along with a codon-optimised mutant and a simple reporter system.

BBa_K322237: firefly luciferase from Photinus pyralis, modified BBa_I712019.

BBa_K322238: firefly luciferase from Photinus pyralis under lac promoter.

BBa_K322451: firefly luciferase from Photinus pyralis, codon optimised and mutated for brighter bioluminescence.

Characterisation


***



References


Fujii, H., Noda, K., Asami, Y., Kuroda, A., Sakata, M. & Tokida, A. (2007). Increase in bioluminescence intensity of firefly luciferase using genetic modification. Analytical Biochemistry 366, 131-136.

Shapirol, E., Lu, C., Baneyx, F. (2009). Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis. Protein Eng Des Sel 22(11): 655-663.

Ljubljana 2007 team wiki, http://parts.mit.edu/igem07/index.php/Ljubljana.




Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.