Part:BBa_K912034:Experience

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Applications of BBa_K912034

Northwestern 2012 iGEM Team: GFP Assay:: To obtain a relative measure of the Pgad activity at varying pH’s of our pH sensor, cells containing the assembled Pgad/GFP nsCP/clc-ec1/xx in pSB1A3 plasmid were grown overnight, diluted 1:500 and then regrown to log phase (all in LB). Once in log phase, the cells were transferred into a 96-well plate with solutions of LB pre-adjusted to various pH levels. Every 5 minutes, the OD and GFP-fluorescence of each well were recorded. The following data were obtained: [http://i.imgur.com/NXS8V.png GFP_assay_results]

Each data point was normalized for LB autofluorescence and for OD (OD shifts were observed based upon the amount of HCl in solution). These abnormalities are seen in the unusually high fluoresence of all wells due to LB autofluoresence and abnormally high OD of the pH 2 wells due to HCl in solution. The results were then normalized again by calculating the fluorescence per OD of cells (pictured below). A t-test was then performed on the two populations of fluoresence/OD numbers, with the null hypothesis stating that the two populations did not have different means and the alternative hypothesis stating that the two populations did have different means. When the t-test was performed, the p-value was 4.9E-009, suggesting that the null hypothesis should be rejected and that there is a statistically significant difference between the mean Fluoresence/OD of the Pgad/GFP + nsCP/clc/xx at pH7 and at pH2. This means that the clc antiporter was activated at low pH conditions, causing chloride ions to flow into the cell, which in turn activated the Pgad promoter, producing the fluorescence found in the assay.

Results of modeling part BBa_K912034: [http://i.imgur.com/8EZbz.png run 1] [http://i.imgur.com/esC0C.png runs 2&3]

Please refer to the results section of our wiki for more information on both this part and related parts http://2012.igem.org/Team:Northwestern/Project/Results

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