Difference between revisions of "Part:BBa K1462090:Design"
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===Design Notes===
 
===Design Notes===
  
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　To test the localization function of CoxVI, the C-terminus of CoxVI was fused to GFP. Besides,we use MitoTracker Red®CMXRos to stain the mitochondria and do an overlap with GFP. By observing the coefficient value, we can judge that if our leading peptide works.
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To test the localization function of CoxVI, the C-terminus of CoxVI was fused to GFP. Besides,we use MitoTracker Red®CMXRos to stain the mitochondria and do an overlap with GFP. By observing the coefficient value, we can judge that if our leading peptide works.<br/>We adopted fluorescence test to have a visible look at where the fusion protein is located. The following microscope images were obtained. It was observed that green fluorescence concentrated in mitochondria stained by a specific stain, instead of diffusing in the cytosol.The reult  proves that CoxVI can locate GFP into the mitochondrial matrix.
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  We adopted fluorescence test to have a visible look at where the fusion protein is located. The following microscope images were obtained. It was observed that green fluorescence concentrated in mitochondria stained by a specific stain, instead of diffusing in the cytosol.The reult  proves that CoxVI can locate GFP into the mitochondrial matrix.
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[[File:G6GT2.jpg|100px|left|frame|Figue1.GFP is targed to the mitochondria]]
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===Source===
 
===Source===
  

Revision as of 07:31, 17 October 2014

Gal1+cox6+GFP+ADH1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1325


Design Notes

To test the localization function of CoxVI, the C-terminus of CoxVI was fused to GFP. Besides,we use MitoTracker Red®CMXRos to stain the mitochondria and do an overlap with GFP. By observing the coefficient value, we can judge that if our leading peptide works.
We adopted fluorescence test to have a visible look at where the fusion protein is located. The following microscope images were obtained. It was observed that green fluorescence concentrated in mitochondria stained by a specific stain, instead of diffusing in the cytosol.The reult proves that CoxVI can locate GFP into the mitochondrial matrix.

Figue1.GFP is targed to the mitochondria

Source

COXVI is obtained from genome of saccharomyces cerevisiae, the other parts is from the kit provided by official

References

Ammy C. Maarse1 et al, Subunit IV of yeast cytochrome c oxidase: cloning and nuicleotide sequencing of the gene and partial amino acid sequencing of the mature protein,The EMBO Journal vol.3 no.12 pp.2831 -2837, 1984