Team:Hong Kong HKUST/characterization/cmv

In our characterization, CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108). The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope. The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator (BBa_K648013) that does not contain the CMV promoter. The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.

Result

Figure 1. MLS directs GFP into mitochondria. When MLS is added to the N terminus of GFP, the GFP was directed to the mitochondria in the cells, giving patches of GFP signal that overlapped with the signals from MitoTracker®. When MLS is not added to the GFP, the GFP signal can be seen scattered all around in the cell. Scale bar = 10 microns

Figure 2. Scatter plots of fluorescence intensities of green (y axis) and red (x axis) from images shown in Figure 1. It showed that the BioBrick MLS-GFP and commercial GFP construct had linear relationship of green intensities and red intensities while the GFP generator had no relationship. Pearson's correlation coefficient (rp) and Spearman correlation coefficient (rs) were determined using the Pearson-Spearman correlation colocalization plugin (French et al., 2008) for ImageJ with a threshold of 0 and listed for each image.

Figure 3. Mean Pearson correlation coefficient(rp) and mean Spearman correlation coefficient(rs) were shown in bar chart. Using ImageJ software and plugins, the Pearson correlation coefficient and Spearman correlation coefficient were generated. For every batch of transfected cells, four samples were used for quantification. Experimental BioBrick MLS-GFP and commercial MLS-GFP: Coefficients were close to 1, good colocalization ; GFP: Coefficients were close to 0, poor colocalization. Error bars show standard deviation.

Conclusion

Reference