Part:BBa_K2557031

miniCMV promoter-Anti-GFP-mnotch-TEV protease-NLS

The Notch receptor is a natural cell surface receptor whose extramembrane domain can be replaced by a variety of receptors, and the intramembrane domain can be replaced with a variety of transcription factors to construct a customized cell signaling pathway. We retained the conserved mmotch transmembrane domain, replacing the extracellular domain with anti-GFP and the intracellular domain with the TEV protease, thereby converting the extracellular signal into an intracellular signal.We inserted the miniCMV promoter upstream so that the components we constructed were expressed at low levels in the cell.

Sequence and Features

Usage and Biology

Cells receive extracellular signals and respond to the signal molecules accordingly. We hope to customize a kind of receptor so that it can recognize the signal molecules and regulate downstream gene expression [1]. We choose synNotch as an ideal receptor.

Verification of the localization of Lag16-synNotch-TEV on cell membrane

Fig. 1 (Left) Fluorescence microscope observation of the cells cross-linked with GFP. (Right) Blank control (without transfection).

Fig. 1 shows that synNotch can be located to the membrane.

Characterization of Lag16-synNotch-TEV

Fig.2. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP. (A) Fluorescence microscope observation of the cells transfected with plasmids containing the gene of cell surface-expressed GFP. (B) Image results developed in Western blot shows that Lag16-SynNotch-TEV affected by surface-expressed GFP can be resolved into FLAG-TEV and V5-mNotch. (C) Gray scale analysis of western blot image shows the relative level of the Flag tagged LaG16-synNotch-TEV affected by cell surface-expressed GFP. Data are mean ±S.E. (n=3). **, p < 0.01; N.S., no significance.

Fig. 2 shows that the modified synNotch can be located on the surface of cell membrane normally and release intracellular domain after receiving external signals. The replacement of intracellular domain with TEV has no effect on the function of synNotch.

Verification of TEV activation transcription system based on modified tetR

Fig. 4 Function verification of TEV elimination of modified tetR inhibition with eukaryotic cells (A)Fluorescence microscope observation of the stably transfferred cell line. (B)Fluorescence microscope observation of the stably transfferred cell line transferred plasmids containing Lag16 - synNotch-TEV and tetO-miniCMV-EGFP genes. (C)Fluorescence microscope observation of the aforementioned Jurkat T cells co-cultured with 293T cells expressing cell surface-expressed GFP for 4 h.

Through fluorescence microscopy, we could observe that the suspended T cells emit green fluorescence, which is clearly distinguished from the weaker green fluorescence of 293T cells expressing surface-expressed GFP deposited at the bottom of the culture medium. The results show that TEV can relieve the inhibition of tetR on the promoter in 293T cells. It means that we have successfully verified the function of TEV - activated transcription system based on the modified tetR in eukaryotic cells.

References

1. Morsut, L., Roybal, K. T., Xiong, X., Gordley, R. M. & Coyle, S. M. Engineering customized cell sensing and response behaviors using synthetic notch receptors. Cell 780–791 (2016). doi:10.1016/j.cell.2016.01.012

2. Circuits, C. A. et al. Precision Tumor Recognition by T Cells With Article Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. 1–10 (2016).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 455
  Illegal PstI site found at 499
  Illegal PstI site found at 818
  
• 12
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  Illegal NheI site found at 62
  Illegal PstI site found at 455
  Illegal PstI site found at 499
  Illegal PstI site found at 818
  Illegal NotI site found at 1616
  
• 21
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• 23
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  Illegal PstI site found at 455
  Illegal PstI site found at 499
  Illegal PstI site found at 818
  
• 25
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  Illegal PstI site found at 455
  Illegal PstI site found at 499
  Illegal PstI site found at 818
  Illegal NgoMIV site found at 254
  Illegal NgoMIV site found at 959
  Illegal NgoMIV site found at 1136
  Illegal NgoMIV site found at 1674
  Illegal AgeI site found at 71
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1295