Difference between revisions of "Team:NAU-CHINA/testh"

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Fig.2. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP.
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Fig4. Function verification of TEV elimination of modified tetR inhibition with eukaryotic cells
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Co-culture the 293T cells expressing GFP on the cell surface with the cells transferred with Lag16-synNotch-TEV for 1h to extract protein for western bolt detection.
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Stably transfer Jurkat T cells with the modified TetR gene to construct a stably transferred cell line. Then transfer plasmids containing Lag16 - synNotch-TEV and tetO-miniCMV-EGFP genes into the aforementioned stably transferred cell. Co-culture the 293T cells expressing GFP on the cell surface with these Jurkat T cells for 4 h when 293T cells were deposited at the bottom of the culture medium and separated from suspended Jurkat T cells.
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(A) Fluorescence microscope observation of the cells  transfected with plasmids containing the gene of cell surface-expressed GFP.
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(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.  
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(A) Fluorescence microscope observation of the stably transfferred cell line.
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(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.  
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Data are mean ±S.E. (n=3).
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(B) Fluorescence microscope observation of the stably transfferred cell line transferred plasmids containing Lag16 - synNotch-TEV and tetO-miniCMV-EGFP genes.
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**, p < 0.01; 
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N.S., no significance.
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(C) Fluorescence microscope observation of the aforementioned Jurkat T cells co-cultured with 293T cells expressing cell surface-expressed GFP for 4 h.
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The above two experiments show 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.
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Eukaryotic verification of TEV activation transcription system based on modified tetR
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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 and the results also confirm preliminarily that our upstream circuit can work normally. However, we have to admit that due to we chosed GFP as our reporter gene, it is difficult to distinguish it from cell surface-expressed GFP. Our verification experiment is not intuitive. If we need to prove the function of TEV suppressing the inhibition of tetR strongly, further optimized experiments are still needed.
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As mentioned earlier, inducible promoters using transcription activator factors that cannot inhibit transcription often have some leakage due to background expression.
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However, our system hopes to realize the absolute function of 0/1 switch, and the background expression is what we do not expect. Therefore, we need to find a transcription activation system with very low background expression.
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Downstream circuit
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Coincidentally, the previous team iGEM 2017 Oxford was making similar attempt. They have designed TEV activation transcription system based on the modified tetR. Although they have not fully proved that the system can work effectively due to time constraints, we believe that their theoretical basis for designing the system is reasonable. Therefore, we attempt to verify their system with eukaryotic cells.
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The downstream pathway is the core circuit for us to realize the threshold function. According to literature [3], they have verified the inversion function of the three recombinases in prokaryotic cells and proved the threshold function of the recombinases, i.e. the recombinases do not have the inversion function at low concentration. Only when the concentration of recombinase reaches a certain threshold, can the recombinases work normally.
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According to the same document, we designed our pathway in eukaryotic cells, expecting to realize threshold switching in eukaryotic cells. For this reason, we try to test the inversion function of recombinases and the threshold characteristics of the combination of three different recombinases ( Bxb1, TP901, φ C31 ) and three promoters with different intensities ( miniCMV, EF1 - α, Ubc ) in eukaryotic cells.
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We also verify the function of RDF [4] to demonstrate our 0/1 switch resettable.
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Functional verification of three kinds of recombinases in HEK 293T cell

Revision as of 08:23, 17 October 2018

Fig4. Function verification of TEV elimination of modified tetR inhibition with eukaryotic cells Stably transfer Jurkat T cells with the modified TetR gene to construct a stably transferred cell line. Then transfer plasmids containing Lag16 - synNotch-TEV and tetO-miniCMV-EGFP genes into the aforementioned stably transferred cell. Co-culture the 293T cells expressing GFP on the cell surface with these Jurkat T cells for 4 h when 293T cells were deposited at the bottom of the culture medium and separated from suspended Jurkat T 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 and the results also confirm preliminarily that our upstream circuit can work normally. However, we have to admit that due to we chosed GFP as our reporter gene, it is difficult to distinguish it from cell surface-expressed GFP. Our verification experiment is not intuitive. If we need to prove the function of TEV suppressing the inhibition of tetR strongly, further optimized experiments are still needed.

Downstream circuit The downstream pathway is the core circuit for us to realize the threshold function. According to literature [3], they have verified the inversion function of the three recombinases in prokaryotic cells and proved the threshold function of the recombinases, i.e. the recombinases do not have the inversion function at low concentration. Only when the concentration of recombinase reaches a certain threshold, can the recombinases work normally.

According to the same document, we designed our pathway in eukaryotic cells, expecting to realize threshold switching in eukaryotic cells. For this reason, we try to test the inversion function of recombinases and the threshold characteristics of the combination of three different recombinases ( Bxb1, TP901, φ C31 ) and three promoters with different intensities ( miniCMV, EF1 - α, Ubc ) in eukaryotic cells.

We also verify the function of RDF [4] to demonstrate our 0/1 switch resettable.

Functional verification of three kinds of recombinases in HEK 293T cell