Part:BBa_K2549022

aCD19-mN1c-GV2

This part is one of our SynNotch receptors, similar to the original one^[1]. αCD19 (Part:BBa_K2549005) is used as the extracellular sensor module to receive the signal input from surface-expressed CD19. 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. GV2 (GAL4-VP64) is a transactivator comprising of a GAL4 DNA binding domain (Part:BBa_K801032) and a tetrameric VP16 transcription activator domain (Part:BBa _K2549045), which is cleaved after SynNotch activation and drives the expression of the amplifier. Besides, a CD8α signal peptide (Part:BBa_K2549044) and a HA tag (Part:BBa_K2549058) are added to the N terminal of αCD19 (Part:BBa_K2549005) 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 XhoI site found at 2009
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 534
Illegal BsaI site found at 1928
Illegal SapI.rc site found at 1408

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 (+Mock), the EGFP (Y axis, driven by GV2 released after SynNotch activation) was low, and it went high only after adding surEGFP but not surCD19.

We did not use αCD19-mN1c-GV2 because of the experiment on the above.

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

↑ 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

[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

[1]