Team:Wisconsin-Madison/ethanol

The primary source of results thus far has been a plate reader. This is a device which can record the optical density (OD – a measure of cell concentration) as well as the fluorescence of up to 96 cultures at once. Ideally, we hope to produce such a robust fluorescent response as to not need the plate reader for qualitative assessments.

Our ethanol sensing system utilizes the arabinose-inducible pBAD promoter to control the transcription of our exaD and exaE genes. In the presence of ethanol, these genes will phosphorylate the PexaA promoter causing the transcription of our RFP (tagRFP, Evrogen).

This graph shows the fluorescent response of cell cultures containing two plasmids: pBAD33bb: exaDE and PexaA: tagRFP in pSB1A2 (pEtRv2.1). The fluorescence of the system at varying ethanol concentrations was normalized to the optical density of their respective cell culture. The cells’ fluorescence was measured over a range of ethanol concentrations from 0-5% EtOH. This ethanol sensing system is “turned on” or induced in the presence of arabinose by the pBAD promoter. The blue data points on the graph represent the fluorescence of cell cultures without any arabinose and therefore the ethanol sensor is uninduced; the red data points represent the fluorescent response of the induced system in the presence of 0.2% arabinose.

From the graph, it can be seen that there is an increase in fluorescence between the induced and uninduced system. This is the result that was expected, however, there isn’t a large difference in fluorescence between the two systems. To further increase this difference in fluorescence, we intend to decrease the “leakiness” of our promoter through directed evolution. Additionally, the graph shows a positive correlation between fluorescence and ethanol concentration; therefore, our ethanol sensor exhibits a linear response in fluorescent intensity with varying ethanol concentrations. The magnitude of the slope of the data points represents the degree of our systems response. Currently, the slope of the data is shallower than desired. We hope to target this issue using a directed evolution construct.