Difference between revisions of "Part:BBa K2549021"
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| − | This part is one of our SynNotch receptors, similar to the original published version<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>. 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, 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. | + | This part is one of our SynNotch receptors, similar to the original published version<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>. 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<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 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. |
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===Biology=== | ===Biology=== | ||
| − | =====It | + | =====Our characterization===== |
| − | + | [[File:bestNotch.jpg|none|400px|thumb|'''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 αCD19-mN1c-tTAA can be significantly activated by surface-expressed CD19. It also has the highest signal-to-noise ratio among all the SynNotch receptors that we submitted. Besides, it has a lower background activation ratio comparing to others. | ||
| + | |||
| + | LaG16-2-mN1c-GV2 ([[Part:BBa_K2549020]]) and αCD19-mN1c-tTAA ([[Part:BBa_K2549021]]) perform well together, which were used as our common Receptors for ENABLE 16 logic gates. | ||
| + | |||
| + | [[Part:BBa_K2549006]] is exactly as the published version, and results are shown below. Our early results demonstrate 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 ; (3) 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. <i>Just use our improved sets, if you want to SynNotch!</i> | ||
| + | |||
| + | [[File:MouseN1ce1c.png|none|333px|thumb|'''Flow cytometry results of the original published SynNotch - not good comparing with ours.''' 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 . ''We used '''50 ng''' SynNotch plasmids for the experiments here, but '''10 ng''' SynNotch plasmids for the experiments above. The later has reduced background activation and increased signal-noise-ratio.'']] | ||
| + | |||
| + | =====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===== | =====SynNotch receptors function well in Morsut L et al 2016===== | ||
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Please refer to the original article for more details. | Please refer to the original article for more details. | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Latest revision as of 02:15, 18 October 2018
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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2493
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 534
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, 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 αCD19-mN1c-tTAA can be significantly activated by surface-expressed CD19. It also has the highest signal-to-noise ratio among all the SynNotch receptors that we submitted. Besides, it has a lower background activation ratio comparing to others.
LaG16-2-mN1c-GV2 (Part:BBa_K2549020) and αCD19-mN1c-tTAA (Part:BBa_K2549021) perform well together, which were used as our common Receptors for ENABLE 16 logic gates.
Part:BBa_K2549006 is exactly as the published version, and results are shown below. Our early results demonstrate 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 ; (3) 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. Just use our improved sets, if you want to SynNotch!
Flow cytometry results of the original published SynNotch - not good comparing with ours. 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 . We used 50 ng SynNotch plasmids for the experiments here, but 10 ng SynNotch plasmids for the experiments above. The later has reduced background activation and increased signal-noise-ratio.
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 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
- ↑ 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