Team:Cambridge/Bioluminescence/G28

From 2010.igem.org

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{{:Team:Cambridge/Templates/rightpic|src=Jungle_book.jpg|300px|right|G28 illuminating the Jungle Book}}
{{:Team:Cambridge/Templates/rightpic|src=Jungle_book.jpg|300px|right|G28 illuminating the Jungle Book}}
{{:Team:Cambridge/Templates/rightpic|src=Space_invader.jpg|400px|right|G28 on a 96 well plate}}
{{:Team:Cambridge/Templates/rightpic|src=Space_invader.jpg|400px|right|G28 on a 96 well plate}}
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James Slock from King's College, PA kindly provided us with plasmids carrying the genes responsible for bioluminescence in ''V. fischeri''. Using Long-Range PCR, we extracted these genes and assembled them into a new operon. As described in the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Background Background section], the lux operon in ''V. fischeri'' is under tight quorum sensing control. In the absence of LuxR protein and AHL the Lux genes are virtually inactive.
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Whilst researching the literature on bioluminescence at the beginning of the summer, we came across a report on [http://departments.kings.edu/biology/lux/bacterial.html 'Molecular Biology Experiments Utilizing the lux Genes of Vibrio fischeri [...]'] by James Slock from King's College, PA. Dr Slock kindly provided us the two plasmids mentioned in these experiments, which carry the complete V. fischeri lux operon (LuxICDABE regulated by Lux R). Using Long-Range PCR, we extracted luxCD, luxAB and luxE individually and assembled them into a new operon. As described in the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Background Background section], lux I and lux R exert tight quorum sensing control on expression of the lux operon in V. fischeri. In the absence of LuxR protein and AHL the Lux genes are virtually inactive.
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In order to relieve this control, we used [https://2010.igem.org/Team:Cambridge/Gibson/Introduction Gibson Assembly] to produce an operon consisting of Lux C, D, A, B, E (in this order, reflecting ''V. fischeri'') under the arabinose-induced promoter pBAD ([http://partsregistry.org/Part:BBa_I0500 BBa_i0500]). We called this construct the LuxBrick. It caused bright and reproducible light output in the transformed E.coli Top10 cells. This new BioBrick ([http://partsregistry.org/Part:BBa_K325909 BBa_K325909]) can be used as an arabinose->light device and is a very useful part if the aim is to get a high bacterial light output. Many of the images in our [https://2010.igem.org/Team:Cambridge/Photos Photo Gallery] were created using Top10 cell transformed with this part.  
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In order to relieve this control, we used [https://2010.igem.org/Team:Cambridge/Gibson/Introduction Gibson Assembly] to generate an operon consisting of Lux C, D, A, B, E (in this order, reflecting V. fischeri) under the arabinose-induced promoter pBAD ([http://partsregistry.org/Part:BBa_I0500 BBa_i0500]). We called this construct the LuxBrick. It caused bright and reproducible light output in the transformed E.coli Top10 cells. This new BioBrick ([http://partsregistry.org/Part:BBa_K325909 BBa_K325909]) can be used as an arabinose->light device and is a very useful part if the aim is to get a high bacterial light output. Many of the images in our [https://2010.igem.org/Team:Cambridge/Photos Photo Gallery] were created using Top10 cell transformed with this part.  
=h-ns mutants=
=h-ns mutants=

Revision as of 21:11, 26 October 2010