Difference between revisions of "Part:BBa K210010:Experience"

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(→‎Applications of BBa_K210010)
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'''iGEM Kyoto 2009'''
 
'''iGEM Kyoto 2009'''
  
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To confirm the function of the signal sequence for importing protein into mitochondria,
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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.  
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we compared the expressioin pattern of sig-GFP or GFP with mitotracker signal.
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GFP signal was detected throughout the GFP expressing cell except for the black spot region in the cytoplasm or in the nuclei, while sig-GFP signal showed the 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.
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The GFP signal was detected throughout the cell except for the black granules in the cytoplasm or the nuclear,
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while sig-GFP signal showed the string-like pattern in the cytoplasm. The black granules in the cytoplasm observed
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in the GFP-expressing cell were stained by mitotracker, and the result indicated that the GFP was
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normally eliminated from mitochondria (Fig.1, GFP and mitotracker merged). In case of sig-GFP,
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mitochondria stained by a mitotracker and the GFP signal showed almost the same pattern.  
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the yellow color in the merge images (Fig. 1, sig-GFP and mitotracker merged) suggested that
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the sig-GFP and mitochondria were colocalized in the HeLa cell.
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We, consequently, our constructed signal sequence has the function of importing protein into mitochondria as expected.
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Revision as of 20:17, 21 October 2009

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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. GFP signal was detected throughout the GFP expressing cell except for the black spot region in the cytoplasm or in the nuclei, while sig-GFP signal showed the 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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