Part:BBa_K216014:Experience

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Applications of BBa_K216014

This part is intended to give a luminescent response to the presence of nitrate or nitrite (see information for parts BBa_K216005, the nitrate/nitrite-responsive promoter, and BBa_J52008, Renilla luciferase, for details of how this works).

User Reviews

Initial Experiments: (Edinburgh iGEM team, 19 Oct 2009) To confirm that this part works, we grew the clone (E. coli JM109/pSB1A2-BBa_K216014) 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 probably not necessary for Renilla luciferase, but we were doing the Photinus luciferase experiments at the same time, so followed the same protocol). To this was then added 2.5 microlitres of 2 mM coelenterazine (Promega S2001, made up by adding 0.295 ml ethanol to the 250 micrograms of coelenterazine in the tube). In a dark room, bioluminescence was clearly visible in the cells which had been grown in the presence of nitrite, but no luminescence was visible in 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, but in qualitative terms, this part appears to work fine.

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