Difference between revisions of "Part:BBa M50011:Experience"

Revision as of 04:14, 12 December 2016

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Please enter how you used this part and how it worked out.

Applications of BBa_M50011

Our first assay was designed to test to efficacy of our promoter. DNA 2.0 provided the rhamnose range for our promoter, which was between 25 µM and 4mM. First, we transformed chemically competent E. coli cells. Then, using a deep 96 well plate, we set up each well to contain our reaction mix, which contained LB, ampicillin, and our transformed cells. However, we varied the concentration of rhamnose for each well, with the concentration increasing per well with the first well containing no rhamnose. We did not add any rhamnose to show that rhamnose was needed in order to see fluorescence. After initial tests, we added another control in our assay. We added another row of E. coli cells (chemically competent without our plasmid) to compare with our transformed cells. These control experiments were also set up on a deep 96 well plate with a separate reaction mix that contained the control cells. Both the transformed and control cells were grow overnight in the deep 96 well plate before being transferred into a shallow 96 well plate. After transferring the cells, we added a consistent concentration and amount of hydrogen peroxide. We allowed the cells to sit with the hydrogen peroxide for a couple of minutes, then we used the plate reader to determine the GFP.

Our second assay was based on a dose response assay for our hydrogen peroxide sensor. It was designed to show that with increased levels of hydrogen peroxide concentration there would be increased fluorescence from the device. Similar to the first assay, we designed a reaction mix, which contained LB, ampicillin, transformed cells, and a rhamnose. We added varying and increasing concentrations of hydrogen peroxide to each well. Also, our negative controls were not exposed to hydrogen peroxide. Similar to our first assay, we added chemically competent E. coli cells an additional negative control to compare to our transformed cells. Those cells were treated the same as the transformed cells, as they were given hydrogen peroxide with varying concentrations. We also created a separate reaction mix for the control experiments with the difference being that the control cells were used in replace of the transformed cells. Similar to the rhamnose assay set up, we added an LB blank, in order to normalize our fluorescence values, incubated the well overnight and added hydrogen peroxide the next day.

User Reviews

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 ===Applications of BBa_M50011===
+Our first assay was designed to test to efficacy of our promoter. DNA 2.0 provided the rhamnose range for our promoter, which was between 25 µM and 4mM. First, we transformed chemically competent E. coli cells. Then, using a deep 96 well plate, we set up each well to contain our reaction mix, which contained LB, ampicillin, and our transformed cells. However, we varied the concentration of rhamnose for each well, with the concentration increasing per well with the first well containing no rhamnose. We did not add any rhamnose to show that rhamnose was needed in order to see fluorescence. After initial tests, we added another control in our assay. We added another row of E. coli cells (chemically competent without our plasmid) to compare with our transformed cells. These control experiments were also set up on a deep 96 well plate with a separate reaction mix that contained the control cells. Both the transformed and control cells were grow overnight in the deep 96 well plate before being transferred into a shallow 96 well plate. After transferring the cells, we added a consistent concentration and amount of hydrogen peroxide. We allowed the cells to sit with the hydrogen peroxide for a couple of minutes, then we used the plate reader to determine the GFP.
+Our second assay was based on a dose response assay for our hydrogen peroxide sensor. It was designed to show that with increased levels of hydrogen peroxide concentration there would be increased fluorescence from the device. Similar to the first assay, we designed a reaction mix, which contained LB, ampicillin, transformed cells, and a rhamnose. We added varying and increasing concentrations of hydrogen peroxide to each well. Also, our negative controls were not exposed to hydrogen peroxide. Similar to our first assay, we added chemically competent E. coli cells an additional negative control to compare to our transformed cells. Those cells were treated the same as the transformed cells, as they were given hydrogen peroxide with varying concentrations. We also created a separate reaction mix for the control experiments with the difference being that the control cells were used in replace of the transformed cells. Similar to the rhamnose assay set up, we added an LB blank, in order to normalize our fluorescence values, incubated the well overnight and added hydrogen peroxide the next day.
 ===User Reviews===