Difference between revisions of "Part:BBa K2549016"
 
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<partinfo>BBa_K2549016 short</partinfo>
 
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This part is one of our SynNotch receptors<ref>Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Morsut L, Roybal KT, Xiong X, ..., Thomson M, Lim WA. Cell, 2016 Feb;164(4):780-91 PMID: 26830878; DOI: 10.1016/j.cell.2016.01.012</ref>. LaG17 ([[Part:BBa_K2549004]]) is used as the extracellular sensor module to receive the signal input from GFP. mN1c ([[Part:BBa_K2549006]]) is served as the transmembrane core domain of SynNotch, which is evident to have a low basal expression and a high activation efficiency. tTAA ([[Part:BBa_K2446057]]) is an improved tetracycline-controlled transcription activator, which is cleaved after SynNotch activation and drives the expression of the amplifier. Besides, a CD8&alpha; signal peptide ([[Part:BBa_K2549044]]) and a Myc-tag ([[Part:BBa_K823036]]) are added to the N terminal of LaG17([[Part:BBa_K2549004]]) for membrane targeting and easy determination of surface expression.
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This part is the first version of our SynNotch receptors, as original published<ref>Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Morsut L, Roybal KT, Xiong X, ..., Thomson M, Lim WA. Cell, 2016 Feb;164(4):780-91 PMID: 26830878; DOI: 10.1016/j.cell.2016.01.012</ref>. LaG17 ([[Part:BBa_K2549004]]) is used as the extracellular sensor module to receive the signal input from GFP. mN1c ([[Part:BBa_K2549006]]) is served as the transmembrane core domain of SynNotch, which is evident to have a low basal expression and a high activation efficiency. tTAA ([[Part:BBa_K2446057]]) is an improved tetracycline-controlled transcription activator<ref>Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Urlinger S, Baron U, Thellmann M, ..., Bujard H, Hillen W. Proc Natl Acad Sci U S A, 2000 Jul;97(14):7963-8  PMID: 10859354</ref>, which is cleaved after SynNotch activation and drives the expression of the amplifier. Besides, a CD8&alpha; signal peptide ([[Part:BBa_K2549044]]) and a Myc-tag ([[Part:BBa_K823036]]) were added to the N terminal of LaG17([[Part:BBa_K2549004]]) for membrane targeting and easy determination of surface expression.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2549016 SequenceAndFeatures</partinfo>
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===Biology===
 
===Biology===
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====SynNotch receptors function well in Lim WA et al in 2016====
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=====Our characterization=====
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[[File:bestNotch.jpg|none|400px|thumb|'''Flow cytometry results of SynNotch activation.''' surAg, surface antigens, which was surface-expressed CD19 for &alpha;CD19-SynNotch or surface-expressed EGFP for LaG-SynNotch, respectively. Without surAg, the EGFP (Y axis, driven by tTAA released after SynNotch activation) was low, and it went high after adding surAg. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Optimization .]]
  
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[[File:SynNotch.jpeg|none|360px|thumb|Lim WA et al stated:''SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.'']]
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It is obvious that LaG17-mN1c-tTAA can be significantly activated by surface-expressed EGFP.
  
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====LaG17-mN1c-tTAA works as expected====
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K2549016 SequenceAndFeatures</partinfo>
 
  
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=====SynNotch receptors function well in Morsut L et al 2016=====
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[[File:SynNotch.jpeg|none|300px|thumb|Morsut L et al stated:''SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.'']]
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[[File:SynNotchECDandICD.jpeg|none|400px|thumb|Morsut L et al have shown that modularity of the synNotch platform. They stated: ''the input and output domains from Notch can be swapped with diverse domains. On the extracellular side, diverse recognition domains can be used (antibody based, or peptide tags are shown) and on the intracellular side, diverse effector can be used (transcriptional activators with different DNA-binding domains are shown, as well as a transcriptional repressor).'']]
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Please refer to the original article for more details.
  
 
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Latest revision as of 19:10, 17 October 2018


LaG17-mN1c-tTAA

This part is the first version of our SynNotch receptors, as original published[1]. LaG17 (Part:BBa_K2549004) is used as the extracellular sensor module to receive the signal input from GFP. mN1c (Part:BBa_K2549006) is served as the transmembrane core domain of SynNotch, which is evident to have a low basal expression and a high activation efficiency. tTAA (Part:BBa_K2446057) is an improved tetracycline-controlled transcription activator[2], which is cleaved after SynNotch activation and drives the expression of the amplifier. Besides, a CD8α signal peptide (Part:BBa_K2549044) and a Myc-tag (Part:BBa_K823036) were added to the N terminal of LaG17(Part:BBa_K2549004) for membrane targeting and easy determination of surface expression.

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 87
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2154
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 127
    Illegal SapI.rc site found at 1069


Biology

Our characterization
Flow cytometry results of SynNotch activation. surAg, surface antigens, which was surface-expressed CD19 for αCD19-SynNotch or surface-expressed EGFP for LaG-SynNotch, respectively. Without surAg, the EGFP (Y axis, driven by tTAA released after SynNotch activation) was low, and it went high after adding surAg. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Optimization .

It is obvious that LaG17-mN1c-tTAA can be significantly activated by surface-expressed EGFP.


SynNotch receptors function well in Morsut L et al 2016
Morsut L et al stated:SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.
Morsut L et al have shown that modularity of the synNotch platform. They stated: the input and output domains from Notch can be swapped with diverse domains. On the extracellular side, diverse recognition domains can be used (antibody based, or peptide tags are shown) and on the intracellular side, diverse effector can be used (transcriptional activators with different DNA-binding domains are shown, as well as a transcriptional repressor).

Please refer to the original article for more details.


References

  1. ↑ Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Morsut L, Roybal KT, Xiong X, ..., Thomson M, Lim WA. Cell, 2016 Feb;164(4):780-91 PMID: 26830878; DOI: 10.1016/j.cell.2016.01.012
  2. ↑ Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Urlinger S, Baron U, Thellmann M, ..., Bujard H, Hillen W. Proc Natl Acad Sci U S A, 2000 Jul;97(14):7963-8 PMID: 10859354