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 Background section, The lux operon in V. fischeri is under tight quorum sensing control. In the absence of LuxR and AHL the Lux genes are virtually inactive. In order to relieve this control, we used 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]).

This construct caused bright and reproducible light output in the transformed E.coli Top10 cells.

h-ns mutants

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.