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Team:Hong Kong HKUST/characterization/mls
From 2013.igem.org
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Mitochondrial Leader Sequence
- Introduction
- Result
- Conclusion
- Reference
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Characterization
- Mitochondrial Leader Sequence
- CMV Promoter
- EF1-alpha Promoter
Mitochondrial Leader Sequence
Introduction
In our characterization, the CDS of MLS was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format(BBa_K648013). The translation unit was driven by CMV promoter (BBa_K1119006) and terminated by hGH polyA signal (BBa_K404108). The MLS-GFP generator (BBa_K1119009) was then transfected into HEK293FT cells. Mitochondria were stained after transfection and co-localization was determined by area of signal that overlapped. To provide a positive control, CDS of EGFP from pEGFP-N1 (Clontech) was inserted downstream and in frame with the CDS of the MLS in the commercial plasmid pCMV/myc/mito, (Invitrogen, Carlsbard, CA). A negative control was made by GFP generator that does not contains the CDS of MLS (BBa_K1119008). 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.
