Part:BBa_K2539300:Hard Information

Construct Design This construct was built to overexpress alcR, a regulatory protein necessary for the activation of PalcA. We acquired all parts from the iGEM distribution kit: a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, strong RBS (BBa_B0034), alcR (BBa_K2092001), and a double terminator (BBa_B0015) to end transcription.

We used green fluorescence protein (GFP) as a reporter to test the function of PalcA. The GFP sequence was placed behind the PalcA promoter, a strong RBS (BBa_B0034), and before a double terminator (BBa_B0015) to express GFP in the presence of ethanol and alcR (PalcA_GFP: BBa_K2539500; Figure 2-10). We then cloned PalcA_GFP (BBa_K2539500) and Pconst_alcR (BBa_K2539300) together onto pSB1C3. All sequences were confirmed by PCR check (Figure 2-10) and Tri-I Biotech.

We tested E. coli carrying Pconst_alcR+PalcA_GFP (BBa_K2539550) using different concentrations of ethanol (Figure 2-11). Bacterial cultures with different plasmids were grown overnight. In addition to Pconst_alcR+PalcA_GFP (BBa_K2539550), cultures of Pconst_alcR (BBa_K2539300), PalcA_GFP (BBa_K2539500), and a positive control expressing GFP were also prepared. An initial fluorescence reading was taken using a 96 well plate reader. Next, different amounts of ethanol were added to each tube, the tubes were sealed to prevent evaporation, and left in a shaking incubator for 24 hours. A final measurement was taken, again using a 96 well plate reader, to detect any changes in fluorescence. If PalcA is functional, then we should see Pconst_alcR+PalcA_GFP (BBa_K2539550) cultures glow green in the presence of ethanol.

Figure 2-11. Relative fluorescence of E. coli cultures carrying Pconst_alcR+PalcA_GFP (BBa_K2539550) with varying amounts of ethanol. Over 24 hours, the sample with 0.3 g ethanol (EtOH) had the biggest increase in fluorescence. Pconst refers to the constitutive promoter we used (BBa_K880005). Lysogeny broth (LB), Pconst_alcR, and PalcA_GFP were used as negative controls for this experiment. (Experiment & Figure: Catherine C, Jake Y, Justin W)

Our results matched the general expected trend (Figure 2-11). Addition of 0.3 g of ethanol to Pconst_alcR+PalcA_GFP (BBa_K2539550) seemed to yield the highest fluorescence, and lower amounts of 0.01 g and 0.1 g ethanol were not significantly different than the controls (LB only, Pconst_alcR, and PalcA_GFP). Over 0.5 g of ethanol seemed to kill the bacteria (3 mL of culture), which matches literature thresholds of ethanol tolerance (Chong et al, 2013). In our tests, 0.3 g of ethanol yielded the greatest amount of GFP; however, this effect was extremely weak and the measured fluorescence was about 500 times lower than the positive control (constitutively expressed GFP). In summary, the ethanol promoter, PalcA, is functional in the presence of alcR and a specific concentration of ethanol, but is very inefficient. Thus, this promoter is not applicable for our goal of regulating ALDH2 production.