Team:UC Chile/Protocols

From 2012.igem.org

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<div id="Luciferase Assay">
<div id="Luciferase Assay">
<h2>Promoters Luciferase Assay</h2>
<h2>Promoters Luciferase Assay</h2>
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We measured:
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(1) Luminiscence of LuxAB genes (from ''Xenorhabdus luminescens'' and ''Vibrio fischeri'') placed under [http://partsregistry.org/Part:BBa_K743003 Pta promoter] in Synechocystis upon induction with decanal and dodecanal. Both testing results at [http://partsregistry.org/Part:BBa_K743014:Experience] and [http://partsregistry.org/Part:BBa_K743015:Experience]
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(2) Luminiscence of [http://partsregistry.org/Part:BBa_K325905 Bacterial Lux reporter
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CDEG pλ AB] upon induction with decanal and dodecanal. Testing results [http://partsregistry.org/Part:BBa_K325905:Experience here]
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Controls used:
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Positive control: E. coli with arabinose-induced [http://partsregistry.org/Part:BBa_K325909 Luxbrick]
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Negative control 1: E. coli wt cells
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Negative control 2: Synechocystis PCC6803 wt cells
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Method
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E. coli and Synechocystis PCC 6803 transformed with the target constructs and negative controls were grown in liquid media at 37 °C (E. coli) and 30 °C (Synechocystis PCC 6803).
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Two hours after the beginning of the experiment, OD at 600 nm was measured for K325905 and Top10 in order to reinoculate at 0.24 OD (600 nm) in a total volume of 1.5 mL.
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Aftewards, LuxBrick was induced with Arabinose to a concentration of 3 mM and left in shaker at 30°C along with K325905 and Top10.
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After two hours, 100 µl of transformed bacteria and negative controls were inoculated in a 96 plate well. Every sample was inoculated in octuplicate. Luminiscence of the wells was measured by illuminometer.
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Several plate measurements were done to the plate with decanal and dodecanal at different concentrations (including 0 concentration).
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Note: This experience was done twice. Measurements were done at 14 and 16 hour of circadian rhythm in order to evaluate production of LuxAB under transaldolase promoter.
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Results at hour 14
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<h1>References</h1>
<h1>References</h1>

Revision as of 01:42, 27 September 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012