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Applications of BBa_K210010

iGEM Kyoto 2009

To confirm the function experimentally of the signal sequence to enable the host protein to pass across the mitochondrial inner membrane, we compared the expression pattern of sig-GFP or GFP to mitotracker signal in HeLa cells. Although the signal sequence is derived from yeast, we determined to use HeLa cell for the experiment since the sequence was expected to work in other eukaryotic cells, and also because yeast cells might be too small to observe its mitochondria.

GFP signal was detected throughout the GFP expressing cell except for the black spot regions in the cytoplasm or in the nuclei, while sig-GFP signal showed string-like pattern in the cytoplasm. Instead, the black spots in the cytoplasm were stained by mitotracker, indicating that GFPs are normally excluded from mitochondria (Fig.1, GFP and mitotracker merged). In sig-GFP expressing cells, on the other hand, GFP signal showed almost the same pattern as mitotracker. The yellow color in the merge images (Fig. 1, sig-GFP and mitotracker merged) suggests that the sig-GFP and mitochondria colocalize in HeLa cells. We, consequently, conclude that our constructed signal sequence can be recognized successfully thus leading its host protein into mitochondria as expectedly.

Figure 1: Confocal microscopic images of GFP or sig-GFP transfected cells. The image lines titled "mitotracker(+)" indicates the samples were stained with mitotracker. The columns showed the mitotracker, GFP, and merged images from left, respectively.

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