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Because we are using pBad as our promoter to start the cascade of responses in our system, we needed to test the leakiness of the promoter. To do this, we put pBad on a 1C3 plasmid with Red Fluorescent Protein (RFP).
The promoter, pBad, turns on in the presence of arabinose and in doing so, allows RFP to be transcribed. Therefore, once arabinose is present, our cells will fluoresce red.
The amount that our cells fluoresce depends on the amount of arabinose. To further characterize this observation, we grew up overnights with varying concentrations of arabinose and measured their fluorescence.
Initially, we documented with pictures to illustrate the change in fluorescence and we observed a relatively smooth increase in brightness as the arabinose concentration increased.
Then we used flow cytometry to more accurately characterize the fluorescence:
As illustrated in the flow cytometry characterization, we do see some cells fluorescing even when no arabinose is present, therefore the pBad promoter is leaky.
Although the promoter is leaky, it is sufficient for our proof of concept at this stage of our project. If the leakiness needed to be addressed, the promoter could be transferred to a lower copy plasmid or one could use a weaker pBAD promoter such as BBa_K206001, thereby reducing the effects of leakiness.
