Difference between revisions of "Part:BBa K511302"

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1005123605 http://manualip2cj.pp.ua/ies1.html http://documentsjgq.pp.ua/bmy2.html http://instructionsqsa.pp.ua/kml2.html http://manualiguj.pp.ua/evr2.html http://manualip2cj.pp.ua/ http://manualiguj.pp.ua/  http://documents140.pp.ua/  http://documents140.pp.ua/rlivwk2.html http://documentsjgq.pp.ua/ http://manualiguj.pp.ua/tfhm1.html      http://instructionsqsa.pp.ua/cfco1.html    http://documents140.pp.ua/coyx1.html  http://documents4xm7.pp.ua/        http://documentsjgq.pp.ua/kuelta1.html                http://documentspacs.pp.ua/                           http://manualip2cj.pp.ua/vnauw2.html
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__NOTOC__
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<partinfo>BBa_K511302 short</partinfo>
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This part encodes a fusion between the Delta-3 ligand and the monomeric mCherry red fluorescent protein.  
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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.
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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.  
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K511302 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K511302 parameters</partinfo>
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==Characterization==
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[[Image:NotchDeltaActivation.png|thumb|left|Figure 1. Activation of Notch-Gal4-ESN by Delta-mCherry in a HEK-293 Sender - CHO Receiver Co-Culture.]]
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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.  
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<html><br><br><br><br><br><br><br><br><br><br>
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<center><b>Timelapse of CHO cells transfected with CMV:tTA and TRE:Delta-mCherry with dox<br>
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<iframe width="420" height="315" src="http://www.youtube.com/embed/oHJhDiTZZCI" frameborder="0" allowfullscreen></iframe></center>
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</b><br>
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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.  
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</html>

Latest revision as of 16:18, 10 May 2013

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:
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  • 12
    INCOMPATIBLE WITH RFC[12]
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    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
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  • 25
    INCOMPATIBLE WITH RFC[25]
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    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
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    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.