Part:BBa_K2549021

aCD19-mN1c-tTAA

This part is one of our SynNotch receptors, similar to the original published version^[1]. It is our favorite. 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. 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 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
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 2493
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 534
Illegal SapI.rc site found at 1408

Biology

Clinical significance of anti-CD19

Please refer to our basic Part:BBa_K2549005 for more details.

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 α CD19-mN1c-tTAA can be significantly activated by surface-expressed CD19. It also has the highest signal-to-noise ratio among all the SynNotch that we submitted. Besides, it has a relatively low wrong activation ratio which can be tolerated. Compared to other SynNotch cells, α CD19 SynNotch cells can be activated more readily by its antigens which means the activation ratio is far ahead than any other SynNotch receptors. Therefore, it becomes our team's favorite!

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]

[2]