Part:BBa_M50011:Experience

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Applications of BBa_M50011

Hypotheses: We hypothesized that the with increased levels of rhamnose, the device would demonstrate increased fluorescence. Similarly, we believed that with increased levels of hydrogen peroxide, there would be increased fluorescence detected.

Methods

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.

Results

Based on the results, we believe that our biological device could indeed detect hydrogen peroxide. The first assay result proved our initial hypothesis that increased levels of rhamnose will lead to increased levels of fluorescence. Additionally, when we compared our control cells to our biological sensor, it showed that our sensor could sense hydrogen peroxide and fluorescence while the control cells did not respond. However, despite these results aligning with our initial hypotheses, the biological device does not seem to function the way that we thought that it would.

With regards to the second assay, because it is known that E. coli naturally generates hydrogen peroxide, and that it struggles to maintain a safe level of the substance, it is a possibility that we damaged the cells and negatively influenced the hydrogen peroxide balance within the cells by adding extracellular hydrogen peroxide. Knowing that E. coli generates its own hydrogen peroxide could also explain why there is fluorescence detected in the negative control-transformed cells that were not exposed to hydrogen peroxide.

Our graphical analysis is demonstrated below:

Stanford Location

Plasmid Name: HyPer DNA 2.0 Gene #: 273927 E. coli Sensor Glycerol stock numbers: 0133027195 & 0133027181

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