Team:WPI Worcester/Results
Results
Overview
We have tested the two eGFP reporters that we designed, and have validated their efficiency. In general, the ATG/ACG reporters have shown high efficiency in terms of the difference in the level of expression between the wild type and mutated version, while the β-globin reporters showed significantly less difference. The exact efficiency of these reporters are shown below:
ATG/ACG eGFP Reporters
We transfected HEK293T cells grown in 6-well plates, fixed the cells to slides, and obtained images via microscopy (details). For each chosen field, two images were obtained in order to perform calculation: one image for green fluorescence (Figure 1a), and one for red fluorescence (Figure 1b). The red fluorescence produced by the constitutively expressed RFP gene (mCherry) serves as an indicator for successful transfection, as well as a baseline for measuring the amount of green fluorescence. The fluorescence was quantified using ImageJ , which measures the amount and intensity of the fluorescence by grey scale.
Figure 1a & 1b: eGFP positive control, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
In Figure 2a, the ATG reporter seems to produce less green fluorescence than the positive control. The amount of red fluorescence is similar in the positive control and the ATG reporter.
Figure 2a & 2b: ATG eGFP reporters, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
In Figure 3a, the only visible cells show a very low level of GFP expression, while most cells cannot be seen. There is a visible difference between Figure 2a and Figure 3a, which suggests that the mutated start codon did have an effect on the translation of the eGFP.
Figure 3a & 3b: ACG eGFP reporters, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
The quantified data indicates relatively high efficiency in the ATG/ACG reporters. According to the microscopy data (Figure 4), the ATG reporter showed 70.57% of fluorescence compared to the positive control, while the ACG reporter only gave 4.21%. Therefore, the mutated start codon is proven to be able to produce the difference between the two versions of the reporter.
Figure 4: Efficiency of ATG/ACG reporters compared to positive control. Error bars represent standard error between normalized results from 3 biological replicates of the experiment. (Standard error <0.05)
The results were further validated using flow cytometry as part of the collaboration with BostonU Wetlab. The results show that the ATG reporter has a similar level of fluorescence compared to the positive control, while the ACG reporter shows much less (7.21%), similar to the microscopy results.
Figure 5: ATG/ACG reporters efficiency confirmation by flow cytometry. The difference between the wild type and mutant reporter is consistent with the microscopy data.
WT/PTC β-globin eGFP Reporters
The validation of the β-globin reporters followed the same procedures as the ATG/ACG reporters.
Figure 6a & 6b: WT β-globin eGFP reporters, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
In Figure 6a, fewer glowing cells are seen, and the fluorescence is visibly weaker. However, the PTC β-globin reporter shows even less expression of the GFP. This difference between the WT and PTC reporters was consistently seen in all replicates of the experiment.
Figure 7a & 7b: PTC β-globin eGFP reporters, HEK293T cells, green/red fluorescence, 100X. The two images are of the same field.
According to the data (Figure 8), the wild type β-globin reporter showed a much lower level of expression (14.77% of positive control) compared to the ATG reporter. Therefore, the difference in the level of expression between the WT and PTC reporter is much smaller, which means lower reporter efficiency. The standard error, however, indicates that the difference that is present is very valid, and the premature termination codon is likely to have an actual impact on the expression of the GFP.
Figure 8: Efficiency of WT/PTC β-globin reporters compared to positive control. Error bars represent standard error between normalized results from 3 biological replicates of the experiment. (Standard error <0.01)
* The complete experiment (RNA editing with APOBEC/ADAR) has been attempted, but no data is available at this point.
Raw Data
The raw data of individual experiments.
