Part:BBa_M36284:Experience

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For this experiment, the actuator was placed in Rhamex 67k Xbrane vector that had a high copy origin of replication, ampicillin resistance, and a rhamnose sensor. The rhamnose sensor induces ADH1B expression in 1000uM rhamnose and represses expression in 0.2% glucose. We carried out lethality tests to evaluate the actuator’s ability to break down ethanol. For the lethality tests, 1mL of E. Coli in LB+amp (OD600 = 0.5) was grown with 2mL of ethanol diluted in dH2O over a range ethanol concentrations. After growing the E. Coli at 37oC overnight, the OD600 was measured again. As a preliminary experiment, we tested ethanol concentrations 50%, 40%, 30%, 20%, 10%, 5%, 3%, 1%, 0.5%, and 0.1% on E. Coli in LB+amp with 0.2% glucose to determine the range for subsequent tests. We tested two sets of E. Coli that were grown overnight with respective inducer and repressor: induced with 1000uM Rhamnose and repressed with 0.2% glucose for our negative control. In both solutions, we added 2ug/mL of zinc sulfate because zinc is a cofactor for ADH1B (UniProt and Yao et al., 2005). After growing the bacteria in various ethanol concentrations overnight at 37oC, we measured and recorded the respective OD’s.

The glucose-repressed negative control that had no expression of ADH1B showed similar OD response to increasing ethanol concentration, as expected. Cell death occurred mostly in the ranges 0-5% and 20-30% ethanol. The rhamnose and glucose curve was very similar to the negative control because of a procedural error where the E. Coli was grown over night with small amounts of glucose ~0.1%. Even though 1000uM rhamnose was added to the solution during the test, the glucose was sufficient to repress ADH1B expression. To assess the functionality of ADH1B, we repeated the lethality test so that E. Coli was grown over night with only rhamnose. The rhamnose only curve in Figure 4 represents the average of 3 trials. Compared to the negative control and rhamnose and glucose curves the rhamnose only curve showed a decrease in growth, which was unexpected because ADH1B breaks down ethanol and allows for E. Coli growth at higher ethanol concentrations. This was probably due to a procedural change where ethanol was added to E. Coli liquid culture and water instead of E. Coli liquid culture added to ethanol diluted in water. Sudden addition of pure ethanol might have resulted in cell death. Over all, the results suggest that our ADH1B actuator is nonfunctional because it was unable to break down ethanol, which would have allowed for E. Coli with induced expression of ADH1B to survive at higher concentrations of ethanol.