Team:Northwestern/Contribution
CHARACTERIZATION
pBAD characterization
The part we chose to characterize was BBa_K584000, a pBad GFP Generator. We wanted to characterize the optimal concentration of arabinose to induce transcription activity of the pBad promoter by measuring the fluorescence per OD of our cells which have a reporter gene, GFP, downstream. To perform this assay, we let E. coli bacteria containing the BBa_K584000 plasmid with GFP grow for 9 hours in a plate reader assay. We inoculated the cultures overnight, diluted them to OD = 0.05 in the morning and we let them grow in triplicates up until OD = 0.1. Based on a previous characterization done, we added in arabinose concentration of 0.1% to 0.5% in increments of 0.1% in order to find the concentration at which pBad promoter is most actively induced.
RESULTS
Through performing our characterization assay, we plotted the growth of fluorescence per OD of our pBad+GFP cells over time (Figure 1). From our data, we were able to determine the saturation point (arabinose = 0.2%) at which the pBad promoter was maximally induced. We additionally created a fold activation bar graph (Figure 2) comparing the fold activation of cells grown in different concentrations of arabinose. We observed through our experiments that the pBad promoter was strongly induced as the arabinose concentration was increased.
LEXA2 DNA DAMAGE SENSOR CHARACTERIZATION
We also provided novel characterization data to part BBa_K079050 (as we refer to it “LexA2”). The 2008 Bologna Team reported their part was unable to be induced by UV light. However, after exposing it to various doses of UV-C (0 J/m^2, 50 J/m^2, and 125 J/m^2), we found that their part showed different responses for different amounts of UV exposure. Specifically, the rate of decrease in Fluorescence/OD is significantly different between each exposure time, providing evidence that this part may actually be able to differentiate between different UV doses, just not in the way it was originally designed for (Figures 3 and 4). Fluorescence measurements were taken every 3 minutes immediately after exposure in a 96-well plate reader for 3 hours.
Figure 3. Fluorescence/OD values for Part BBa_K079050 after exposure to various UV doses.
Figure 4. Fold Activation for part Part BBa_K079050 after exposure to various UV doses.
Figure 3. Fluorescence/OD values for Part BBa_K079050 after exposure to various UV doses.
Figure 4. Fold Activation for part Part BBa_K079050 after exposure to various UV doses.