Part:BBa_K216015:Experience

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Initial Experiments: (Edinburgh iGEM team, 19 Oct 2009) To confirm that this part works, we grew the clone (E. coli JM109/pSB1A2-BBa_K216015) overnight in LB with shaking, in the presence of 80 mg/l ampicillin, with and without 30 mM sodium nitrite (which was found to be the best inducer for the yeaR promoter; see notes for BBa_K216005). The following day, cells from 1 ml were spun down and resuspended in 0.5 ml 5 mM citrate buffer, pH 4.8 (this is reported to increase luminescence in whole cells by a factor of around 50 compared to using neutral medium, presumably by changing the ionization state of the luciferin so that it permeates cells better). To this was then added 5 microlitres of 10 mM D-luciferin, sodium salt (Promega E1601, made up by adding 1.57 ml 10 mM Tris buffer, pH 8, to the 5 mg of luciferin in the bottle). In a dark room, faint bioluminescence was visible in both tubes, ie both the cells which had been grown in the presence of nitrite, and the cells which had been grown in LB without nitrite. Unfortunately, at the time of writing we don't have access to a luminometer to quantify the luminescence. Further experiments are needed to confirm inducibility of this construct, but there is definitely some light produced, which is good, considering the extra DNA between the RBS and coding sequence (see Design page).

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