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Part:BBa_K1119009

Designed by: Hyun Jung Lee   Group: iGEM13_Hong_Kong_HKUST   (2013-09-16)

CMV promoter - MLS - GFP - hGH polyA tail

In characterization of Mitochondrial Leader Sequence(MLS) (BBa_K1119001), 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). This construct was used for characterization of MLS BioBrick(BBa_K1119001) .

Result

This MLS-GFP generator (BBa_K1119009) was 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).

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. Scale bar = 10 microns

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 628
    Illegal AgeI site found at 1462
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1865
    Illegal BsaI.rc site found at 1367


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Categories
//chassis/eukaryote/human
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