Composite

Part:BBa_K2549017

Designed by: Rongrong Du   Group: iGEM18_Fudan   (2018-10-09)


LaG17-mN1ce-tTAA

This part is the one original published[1], and we test but not used for further experiments. LaG17 (Part:BBa_K2549004) is used as the extracellular sensor module to receive the signal input from GFP. mN1ce (Part:BBa_K2549007), which has 3 more EGF repeats than mN1c, is served as the transmembrane core domain of SynNotch. tTAA (Part:BBa_K2446057) is an improved tetracycline-controlled transactivator[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) are 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 2265
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 127
    Illegal SapI.rc site found at 1180


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 .

Please note that LaG17-mN1ce-tTAA was not test in the experiment above.

Flow cytometry results of the original published SynNotch. surEGFP is the surface expressed EGFP, which was used as the antigen. Without antigen (+Mock), the EGFP (Y axis, driven by tTAA released after SynNotch activation) was low, and it went "high" after adding the antigen. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Optimization .

Part:BBa_K2549006 used in the experiments above is exactly as the published version, and the only difference was we transiently transfected the cells while the authors created stable cells and picked a cell clone for further experiments. Our results show three things: (1) SynNotch background activation is very high, which is why we put a lot effect in http://2018.igem.org/Team:Fudan/Optimization ; (2) mN1c might be better than mN1ce, not only shorter but a little higher signal-noise-ratio; (3) LaG17 is NOT a good extracellular antibody for SynNotch.

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
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Categories
//cds/membrane
//cds/receptor
Parameters
None