Difference between revisions of "Part:BBa K2549061"

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<partinfo>BBa_K2549061 short</partinfo>
 
<partinfo>BBa_K2549061 short</partinfo>
  
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This part contains our SynNotch receptor. The extracellular domain of SynNotch is LaG16 which is a nanobody with high affinity and high specificity to EGFP. Mouse Notch1 extended core region is served as the Notch core. tTA is a transcription activator. A CD8&alpha; signal peptide is placed on the N terminal to the SynNotch receptor for membrane localization. V5 tag is fused to the SynNotch receptor for easy determination of surface expression. A CMV promotor which can conduct a strong constitutive expression is set before SynNotch. A P2A-EGFP is placed into the same open reading framework with SynNotch to ensure its expression.
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This part contains our SynNotch receptor. The extracellular domain of SynNotch is LaG16 which is a nanobody with high affinity and high specificity to EGFP. Mouse Notch1 extended core region is served as the Notch core. tTA is a transcription activator. A CD8&alpha; signal peptide is placed on the N terminal to the SynNotch receptor for membrane localization. V5 tag is fused to the SynNotch receptor for easy determination of surface expression. A CMV promotor which can conduct a strong constitutive expression is set before SynNotch. A P2A-EGFP is placed into the same open reading framework with SynNotch to ensure its expression. This part is copied from a viral vector which can be expressed in mammalian cells.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2549061 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2549061 SequenceAndFeatures</partinfo>
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<!-- Add more about the biology of this part here -->
 
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===Characterization===
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===Our characterization===
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We have tested our SynNotch using surface-expressed EGFP. It is shown that our SynNotch receptor performs a contact-dependent behaviour when co-culturing with surEGFP.
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Please refer to our previous wiki http://2017.igem.org/Team:Fudan/Demonstrate for more experimental details. Last year, we have tested this SynNotch using surface-expressed EGFP.
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[[File:antigen-surEGFP.jpeg|none|720px|thumb|'''SynNotch receptor shows an antigen-dependent behaviour.''' We used fluorescence confocal microscopy to capture images for this figure. maxZ is the max intensity projection of all slices (about 80 in total) at a given position, while slice<sup>51</sup> was a middle bottom optical slice. OE means overexposed images to reveal details. EGFP signals in green is the antigen, and mCherry signal in red is the output. We present this data in 2017 iGEM. And, this plasmid has been requested by others since then.]]
  
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[[File:antigen-surEGFP.jpeg|none|360px|thumb|'''Fig 1. Our SynNotch receptor shows an antigen-dependent behaviour. We used fluorescence microscopy to capture this figure.''']]
 
  
 
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Latest revision as of 00:36, 18 October 2018


CMV-CD8α-V5-LaG16-mN1ce-tTA-P2A-EGFP

This part contains our SynNotch receptor. The extracellular domain of SynNotch is LaG16 which is a nanobody with high affinity and high specificity to EGFP. Mouse Notch1 extended core region is served as the Notch core. tTA is a transcription activator. A CD8α signal peptide is placed on the N terminal to the SynNotch receptor for membrane localization. V5 tag is fused to the SynNotch receptor for easy determination of surface expression. A CMV promotor which can conduct a strong constitutive expression is set before SynNotch. A P2A-EGFP is placed into the same open reading framework with SynNotch to ensure its expression. This part is copied from a viral vector which can be expressed in mammalian cells.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3156
    Illegal XbaI site found at 2154
    Illegal PstI site found at 924
    Illegal PstI site found at 968
    Illegal PstI site found at 1287
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3156
    Illegal NheI site found at 531
    Illegal PstI site found at 924
    Illegal PstI site found at 968
    Illegal PstI site found at 1287
    Illegal NotI site found at 2085
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3156
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3156
    Illegal XbaI site found at 2154
    Illegal PstI site found at 924
    Illegal PstI site found at 968
    Illegal PstI site found at 1287
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3156
    Illegal XbaI site found at 2154
    Illegal PstI site found at 924
    Illegal PstI site found at 968
    Illegal PstI site found at 1287
    Illegal NgoMIV site found at 723
    Illegal NgoMIV site found at 1428
    Illegal NgoMIV site found at 1605
    Illegal NgoMIV site found at 2143
    Illegal NgoMIV site found at 3198
    Illegal AgeI site found at 540
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1764


Our characterization

Please refer to our previous wiki http://2017.igem.org/Team:Fudan/Demonstrate for more experimental details. Last year, we have tested this SynNotch using surface-expressed EGFP.

SynNotch receptor shows an antigen-dependent behaviour. We used fluorescence confocal microscopy to capture images for this figure. maxZ is the max intensity projection of all slices (about 80 in total) at a given position, while slice51 was a middle bottom optical slice. OE means overexposed images to reveal details. EGFP signals in green is the antigen, and mCherry signal in red is the output. We present this data in 2017 iGEM. And, this plasmid has been requested by others since then.