Part:BBa_K2549019

LaG16-2-mN1c-tTAA

This part is the second version of our SynNotch receptors, as original published^[1]. LaG16-2 (Part:BBa_K2446058) 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 transactivator^[2], which is cleaved after SynNotch activation and drives the expression of the amplifier.

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 381
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 2580
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1495

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 LaG16-2-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.

Characterization

It works as we designed.

Flow cytometry results of SynNotch receptors. surAg, surface antigens, which represent surface-expressed CD19 or surface-expressed EGFP, respectively. A EGFP-P2A circuit is placed downstream the SynNotch, which means they are in the same open reading framework.

It is obvious that LaG16-2-mN1c-tTAA can be significantly activated by surface-expressed EGFP.

References

↑ 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
↑ 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

[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

[1]

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