Part:BBa_K1119001

Mitochondrial Leader Sequence with RFC25 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. The CDS of MLS was cloned out from pCMV/myc/mito (Invitrogen, Carlsbard, CA) using PCR. 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. 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.

This part is in RFC25 standard to facilitate fusing with other CDS. MLS in RFC10 standard (BBa_K1119000)is submitted as alternative. but cannot be fused directly to other CDS due to limitations in RFC10. Users who obtained the part in RFC10 standard can obtain the part by PCR and fuse to other domains using Splicing by Overlapping PCR.

Characterization

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 [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization detailed protocol] of our characterization can be found in HKUST iGEM 2013 Wiki.

Figure 1. MLS directs GFP into mitochondria. When MLS is added to the N terminus of GFP, the GFP was directed to the mitochondria in the cells, giving patches of GFP signal that overlapped with the signals from MitoTracker®. When MLS is not added to the GFP, the GFP signal can be seen scattered all around in the cell. The contrast for the negative control is up tuned since the raw signal was too dim to be visible. Scale bar = 10 microns

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]