Team:Cambridge/Bioluminescence/G28

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=h-ns mutants=
=h-ns mutants=
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To test the theory that H-NS proteins repress the Lux genes by interacting with their coding regions, we transformed two E.coli mutant strains with this construct and measured their light output. The strains we used were Gm230 hns205::kan, which has a C-terminal deletion in the H-NS gene and MC4110 delta hns::tet, a strain from a knockout library. In the literature, h-ns mutant strains have been described as producing much brighter luminescence than wild type strains. While we could not reproduce a higher peak brightness, it was apparent that the knockout strain maintained its light output for much longer than wild type, which showed a steep reduction in brightness upon entering stationary phase. This phenomenon (Abrupt Decline in Luciferase Activity - or ADLA) has been described by [http://www.springerlink.com/content/w73k840k27866462/fulltext.pdf Koga et al. 2004]. This observation gives evidence, that H-NS proteins are involved in regulating the lux operon irrespective of the promoter.
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To test the theory that H-NS proteins repress the Lux genes by interacting with their coding regions, we transformed two E.coli mutant strains with this construct and measured their light output. The strains we used were Gm230 hns205::kan, which has a C-terminal deletion in the H-NS gene and MC4110 delta hns::tet, a strain from a knockout library. In the literature, h-ns mutant strains have been described as producing much brighter luminescence than wild type strains. While we could not reproduce a higher peak brightness, it was apparent that the knockout strain maintained its light output for much longer than wild type, which showed a steep reduction in brightness upon entering stationary phase. This phenomenon (Abrupt Decline in Luciferase Activity - or ADLA) has been described by [http://www.springerlink.com/content/w73k840k27866462/fulltext.pdf Koga et al. 2004]. However, this paper suggests that the effect of H-NS on luminescence is not due to expression of the lux genes, but occurs indirectly via the cell's redox pool, in particular the availability of FMNH2.
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Revision as of 14:27, 25 October 2010