Team:Hong Kong HKUST/characterization/mls
From 2013.igem.org
![](https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png)
-
Mitochondrial Leader Sequence
- Introduction
- Result
- Conclusion
- Reference
-
Characterization
- Mitochondrial Leader Sequence
- CMV Promoter
- EF1-alpha Promoter
Mitochondrial Leader Sequence
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
![](https://static.igem.org/mediawiki/parts/b/b9/Mlschar_1.jpg)
![](https://static.igem.org/mediawiki/parts/8/80/Scatterplots_mlsquantification.jpg)
![](https://static.igem.org/mediawiki/parts/c/c2/Barchart_mlsquantification.jpg)