Part:BBa_K511302

Delta-mCherry Juxtacrine Signaling Ligand MammoBlock

This part encodes a fusion between the Delta-3 ligand and the monomeric mCherry red fluorescent protein.

In complex eukaryotic systems, Delta is a single-pass transmembrane protein that functions as a ligand for another single-pass receptor known as Notch. Binding of Delta to Notch results in cleavage of the intracellular domain of Notch by a ubiquitously expressed protease. The cleaved intracellular domain is then translocated to the nucleus of the Notch-bearing cell and is able to activate transcription of target genes. If Delta and Notch are expressed on the same cell, however, Delta binds to a second location on the Notch receptor, forming a complex that cannot be activated by Delta from another cell.

The C-terminal fusion of the mCherry protein to this signaling ligand means this protein is also a useful marker for transfection identification and/or membrane colocalization experiments.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 85
  Illegal PstI site found at 162
  Illegal PstI site found at 1005
  Illegal PstI site found at 1428
  Illegal PstI site found at 2105
  Illegal PstI site found at 2491
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 85
  Illegal PstI site found at 162
  Illegal PstI site found at 1005
  Illegal PstI site found at 1428
  Illegal PstI site found at 2105
  Illegal PstI site found at 2491
  Illegal NotI site found at 494
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 85
  Illegal PstI site found at 162
  Illegal PstI site found at 1005
  Illegal PstI site found at 1428
  Illegal PstI site found at 2105
  Illegal PstI site found at 2491
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 85
  Illegal PstI site found at 162
  Illegal PstI site found at 1005
  Illegal PstI site found at 1428
  Illegal PstI site found at 2105
  Illegal PstI site found at 2491
  Illegal AgeI site found at 521
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 33
  Illegal BsaI site found at 473
  Illegal BsaI.rc site found at 1738
  Illegal BsaI.rc site found at 1910
  Illegal SapI site found at 1226
  Illegal SapI.rc site found at 1998
  

Characterization

Figure 1. Activation of Notch-Gal4-ESN by Delta-mCherry in a HEK-293 Sender - CHO Receiver Co-Culture.

Figure 1 to the left shows activation of chinese hamster ovary (CHO) cells bearing Notch-Gal4-ESN driven by the CMV promoter and Citrine driven by the UAS-Gal4 promoter by human embryonic kidney (HEK-293) cells bearing Delta-mCherry driven by the Hef1a-LacO promoter. The images is an overlay of yellow fluorescence from Citrine and red fluorescence from Delta-mCherry. The abnormal morphology of red-fluorescence here was likely due to bleed-through, thus making cells appear larger than they are.

Timelapse of CHO cells transfected with CMV:tTA and TRE:Delta-mCherry with dox

The video is a timelapse of CHO cells transfected with CMV:tTA and TRE:Delta-mCherry. At t=0, 100ug/mL dox is added to the media. The movie is a timelapse sped up such that one second of movie time is one hour of real time.