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Team:Northwestern/Results/Fluorescence
From 2013.igem.org
Single State Characterization
Fluorescence Assay
Due to time constraint, only single state promoters were fully characterized. However, it will be ready for the final presentation. The objective of this assay is to determine the activity of the promoters under a range of pH conditions, ranging from 3.5 to 7.5. Each construct was done in triplicate for each pH. See the protocol section for a detailed protocol followed.
The plate reader results echoed the results found in the overnight tube tests in that the maximum optical density increases as pH increases. In fact, both pH 3.5 and 4.5 both had negligible growth. Despite the difference in the maximum optical densities, all three constructs, Lpp-RBS-GFP (LRG), GadA-RBS-GFP (GRG), and Asr-RBS-GFP (ARG), all had the same growth rate, independent of the pH. This suggests that the pH had no effect on the bacteria’s ability to grow. Instead, the more acidic conditions force the bacteria to stop growth earlier because it faces pH limited growth.
The fluorescence of data confirmed the ASR promoter as an acidic promoter. It only activates for pH 5.5 with only basal level of fluorescence for all other pH. However, GadA does not seem to have the same level of pH control. Increasing pH corresponds to increasing fluorescence levels. This characterization presents a different result that the data from the 2010 University of Wisconsin iGEM team. The wider range of pH in this assay also shows a fuller picture of how the promoter behaves. The Lpp promoter behaves as expected. It has the highest amount of GFP than either of the acidic responsive promoters in all pH ranges. It also shows little signs of any pH responsive element to this constitutive promoter.
Normalized Data
Since we are ultimately interested in the promoter activity, the data needs to be normalized to see how much GFP each cell is producing. In addition, due to the limitations of the plate reader, the background noise also needs to be accounted for in the data. To do this, the background media noise, in the form of a non-zero absorbance 600 value and fluorescence value, is subtracted from each data point, and the background-adjusted fluorescence data is then divided by the optical density values to obtain the fluorescence per optical density reading. This value is proportional to the amount of GFP per cell which is ultimately what correlates to the promoter activity.
The normalized fluorescence data show an interesting result. The general trend for both LRG and GRG is that the the fluorescence per optical density does not differ significantly in different pH range. Since only cells under pH of 5.5, 6.5, and 7.5 grew, the erratic data for cells growing under pH of 3.5 and 4.5 mean very little and can be attributed to the lower limits of the plate reader instrument. While the LRG’s independence of pH is expected, the similarity of GRG’s fluorescence per optical density for pH 5.5 - 7.5 is very surprising. This normalized data is a result of the curves discussed above and further affirm the conclusions made previously.
In contrast to GRG, ARG shows a very strong separation for pH 5.5 versus the rest of the pH range. While the normalized fluorescence is relatively low for all other pH, the normalized fluorescence for pH 5.5 is significantly higher, showing that the promoter is indeed turning on at this pH, again affirming the conclusions made above.
