Difference between revisions of "Part:BBa K1119000"
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Mitochondrial Leader Sequence (MLS) helps direct protein to the mitochondria when this peptide sequence is in front of the N-terminus of the protein of interest. MLS will be removed upon the peptide’s translocation into the mitochondria, but four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein. (Invitrogen, Carlsbard, CA) Under the RFC25 standard, two additional amino residue linker (Ala-Gly) will be left between the MLS remnant and N-terminus of the protein of interest. | Mitochondrial Leader Sequence (MLS) helps direct protein to the mitochondria when this peptide sequence is in front of the N-terminus of the protein of interest. MLS will be removed upon the peptide’s translocation into the mitochondria, but four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein. (Invitrogen, Carlsbard, CA) Under the RFC25 standard, two additional amino residue linker (Ala-Gly) will be left between the MLS remnant and N-terminus of the protein of interest. | ||
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| + | ===Characterization=== | ||
In our characterization, the CDS of MLS was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format. The translation unit was driven by CMV promoter () and terminated by hGH polyA signal (). | In our characterization, the CDS of MLS was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format. The translation unit was driven by CMV promoter () and terminated by hGH polyA signal (). | ||
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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. | 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. | ||
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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). | 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). | ||
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The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki. | The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki. | ||
Revision as of 05:03, 26 September 2013
Mitochondrial Leader Sequence in RFC10 standard
Mitochondrial Leader Sequence (MLS) helps direct protein to the mitochondria when this peptide sequence is in front of the N-terminus of the protein of interest. MLS will be removed upon the peptide’s translocation into the mitochondria, but four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein. (Invitrogen, Carlsbard, CA) Under the RFC25 standard, two additional amino residue linker (Ala-Gly) will be left between the MLS remnant and N-terminus of the protein of interest.
Characterization
In our characterization, the CDS of MLS was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format. The translation unit was driven by CMV promoter () and terminated by hGH polyA signal ().
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]