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

Revision as of 08:19, 17 October 2018

Fig.2. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP. 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. (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. 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.

Eukaryotic verification of TEV activation transcription system based on modified tetR As mentioned earlier, inducible promoters using transcription activator factors that cannot inhibit transcription often have some leakage due to background expression.

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.

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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-Overview
+Fig.2. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP.
-Due to the complex circuit design of our subject, the numerous combination of promoters and gene elements lead to the effect on the whole system in case of the malfunction of any parts, making it difficult for us to locate the malfunction. Therefore, we decided to adopt the method of debugging the program usually employed by computer programmers.
+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.
+(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.
+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.
-)Verify the function of each part.
+Eukaryotic verification of TEV activation transcription system based on modified tetR
+As mentioned earlier, inducible promoters using transcription activator factors that cannot inhibit transcription often have some leakage due to background expression.
-)Combine the parts into two large modules of upstream and downstream circuits to verify the function.
+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.
-)Assemble the upstream and downstream circuits to verify the function of the whole system.
+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.
-However, due to time constraints, we cannot complete such detailed and complete functional verification of various combination designs in just a few months. We have completed the functional verification for most parts and some of the upstream and downstream circuits, but time does not allow us to combine the upstream and downstream circuits for final functional verification. This is undoubtedly regretful, but we have provided concrete ideas for future experiments to help us complete the improvement of the subject. We also put these future experiments on our Wiki.
-Demonstrate
-Upstream circuit
-The upstream circuit mainly designs a signal path to enable cells to receive specific external signals and activate the downstream core circuit to realize the threshold function. Therefore, the upstream circuit can be replaced considering different situations. Here, we provide an upstream circuit design as a reference and other researchers can design their own upstream path to their own needs.
-Customizing the signal path of cells in response to external signals
-There is a wide type of extracellular signals. 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.
-Similar to some signal molecules, take Epidermal Growth Factor Receptor in our realistic system as example, the GFP is also protein but more stable and has no impact on the system. As for visibility and operability, cell surface-expressed GFP as a model of extracellular signal molecule is a better choice. Therefore, we want to replace the excellular domain of synNotch with Lag16, a kind of antigen of GFP. Similar parts have been used in previous project (iGEM 2017 Fudan). We received the plasmids with the gene of cell surface-expressed GFP (标注复旦Part名） and synNotch (标注复旦Part名） from iGEM 2018 Fudan team. But the intracellular domain of synNotch is tTA, a kind of activation factor. Since synNotch was applied to the transformation of cells, the intracellular domain has been replaced by transcription activator factors such as GAL - VP64 and tTA [2]. However, promoters are not completely non-expressed until they are activated, and they often have background expression. Moreover, we hope to make some changes to the intracellular domain of synNotch, trying to replace the intracellular domain with non-traditional transcriptional activator factor to broaden the selection and application of synNotch intracellular domain.
-We found that the previous team iGEM 2017 Oxford modified tetR by replacing the domain between tetR DNA binding domain and regulatory core domain with TEV enzyme cleavage site, so that tetR will be destroyed in the presence of TEV, losing the function of repressing promoter after tetO sequence and opening up the expression of downstream genes.
-According to the idea of iGEM 2017 Oxford, we replaced the intracellular domain of synNotch with TEV and repeated the function verification of synNotch to explore whether replacing intracellular domain with TEV will affect the function of synNotch.