Difference between revisions of "Part:BBa K216015:Experience"

Revision as of 16:47, 20 October 2009

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K216015

This part is designed to produce a luminescent signal in the presence of nitrite or nitrate. See notes on the promoter, BBa_K216005, and firefly luciferase, BBa_I712019, for more information.

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_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; Kobotake et al, 1995, Biosensing of benzene derivatives in the environment by luminescent Escherichia coli. Biosensors & Bioelectronics 10, 601-605). 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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 ===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_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).
+'''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; Kobotake et al, 1995, Biosensing of benzene derivatives in the environment by luminescent ''Escherichia coli. Biosensors & Bioelectronics'' '''10''', 601-605). 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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