Part:BBa_K1119009

CMV promoter - MLS - GFP - hGH polyA tail

The CDS of MLS (BBa_K1119001) 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. 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
  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