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

Revision as of 08:38, 17 October 2018

Function verification of RDF in HEK 293T cell Fig.7. Function verification of the reversal ability of recombinase-RDFs in HEK 293 T Cells Fluorescence microscope observation of HEK 293 T transfect with plasmids containing the recombinase-RDF recognition sites and corresponding recombinase-RDF genes. The image under fluorescence microscope for 293T cells, transfected with plasmids containing the recombinase recognition sites (upper panel) or transfected with plasmids containing corresponding recombinase gene together (bottom panel), are shown.

The results show that the recombinase-RDFs can recognise the sites and reverse the sequence between sites in HEK 293T.

Conclusion We verified the functions of most parts and most upstream and downstream paths step by step. We verified the function of synNotch, the inhibition of tetR after modification, the reversal function and threshold characteristics of some recombinase and promoter combinations. However, due to time constraints, we are unable to complete verification of TEV and the combinations of some recombinases and promoters. Moreover, the combination of upstream and downstream circuits needs to be verified by experiments. We will carry out supplementary experiments in the future to carry out a complete experimental verification of our subject. Future experiments In a short period of one year, it is not easy to fully realize such a complex idea. Therefore, we have envisaged the next series of experiments to further realize our subject idea, combining the idea of continuous feedback between modeling and wet lab to ensure the best system. 1. Optimized function verification of TEV suppressing tetR Inhibition As mentioned earlier, since the reporter gene selected GFP, our experimental results are not intuitive. We will replace the reporter gene with RFP to solve this problem. 2. Verification of the combinations of remaining recombinases and promoters We plan to continue the experiment of remaining combinations that have not yet been verified in order to verify the function and threshold characteristics of these combinations of recombinases and compare the inversion efficiency of recombinases.

3. Construction of a fully functional stable cell line combining upstream and downstream circuits

We plan to finally construct our parts on two plasmids.  Stable cell lines with complete functions were constructed through Puro and BSD screening and their concentration threshold functions will be verified by using agarose beads with different amounts of GFP adsorbed. We intend to apply it to real life.

4. Upgrade our system

The above mentioned is only a condensed version of our ultimate system which include inhabitor and more efficient RDF. We hope to upgrade the condensed version to the final version, which also requires the search for appropriate inhabitor and more efficient RDF. We look forward to the day when our final version will come into being.

Reference [1] 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). [2] 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 [3] Rubens, J. R., Selvaggio, G. & Lu, T. K. Synthetic mixed-signal computation in living cells. Nat. Commun. 7, 1–10 (2016). [4] Rutherford, K. & Van Duyne, G. D. The ins and outs of serine integrase site-specific recombination. Curr. Opin. Struct. Biol. 24, 125–131 (2014).

@@ Line 1: / Line 1: @@
-Fig.5. Function verification of recombinases in  HEK 293T cell
+Function verification of RDF in HEK 293T cell
-Fluorescence microscope observation of HEK 293 T transfect with plasmids containing the recombinase recognition sites and corresponding recombinase gene.
+Fig.7. Function verification of the reversal ability of recombinase-RDFs in HEK 293 T Cells
-The image under fluorescence microscope for 293T cells, transfected with plasmids containing the recombinase recognition sites (the first column picture) or transfected with plasmids containing corresponding combination of promoters and recombinase genes (other column pictures) together, are shown.
+Fluorescence microscope observation of HEK 293 T transfect with plasmids containing the recombinase-RDF recognition sites and corresponding recombinase-RDF genes. The image under fluorescence microscope for 293T cells, transfected with plasmids containing the recombinase recognition sites (upper panel) or transfected with plasmids containing corresponding recombinase gene together (bottom panel), are shown.
-  The results show that the recombinases can recognise the sites and reverse the sequence between sites in HEK 293T.
+  The results show that the recombinase-RDFs can recognise the sites and reverse the sequence between sites in HEK 293T.
-Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells
+Conclusion
+We verified the functions of most parts and most upstream and downstream paths step by step. We verified the function of synNotch, the inhibition of tetR after modification, the reversal function and threshold characteristics of some recombinase and promoter combinations. However, due to time constraints, we are unable to complete verification of TEV and the combinations of some recombinases and promoters. Moreover, the combination of upstream and downstream circuits needs to be verified by experiments. We will carry out supplementary experiments in the future to carry out a complete experimental verification of our subject.
+Future experiments
+In a short period of one year, it is not easy to fully realize such a complex idea. Therefore, we have envisaged the next series of experiments to further realize our subject idea, combining the idea of continuous feedback between modeling and wet lab to ensure the best system.
+. Optimized function verification of TEV suppressing tetR Inhibition
+As mentioned earlier, since the reporter gene selected GFP, our experimental results are not intuitive. We will replace the reporter gene with RFP to solve this problem.
+. Verification of the combinations of remaining recombinases and promoters
+We plan to continue the experiment of remaining combinations that have not yet been verified in order to verify the function and threshold characteristics of these combinations of recombinases and compare the inversion efficiency of recombinases.
+. Construction of a fully functional stable cell line combining upstream and downstream circuits
+ We plan to finally construct our parts on two plasmids.  Stable cell lines with complete functions were constructed through Puro and BSD screening and their concentration threshold functions will be verified by using agarose beads with different amounts of GFP adsorbed. We intend to apply it to real life.
+. Upgrade our system
+  The above mentioned is only a condensed version of our ultimate system which include inhabitor and more efficient RDF. We hope to upgrade the condensed version to the final version, which also requires the search for appropriate inhabitor and more efficient RDF. We look forward to the day when our final version will come into being.
+Reference
+[1] 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).
+[2] 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
+[3] Rubens, J. R., Selvaggio, G. & Lu, T. K. Synthetic mixed-signal computation in living cells. Nat. Commun. 7, 1–10 (2016).
-Fig.6. Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells
+[4] Rutherford, K. & Van Duyne, G. D. The ins and outs of serine integrase site-specific recombination. Curr. Opin. Struct. Biol. 24, 125–131 (2014).
-HEK 293T cells were co-transfected with six different amounts of plasmids containing recombinase genes (miniCMV-Bxb1 and miniCMV-TP901) , and fixed numbers of plasmids containing corresponding recombinase recognition sites. After 36 hours of plasmid co-transfection, the proportion of fluorescent cells and the average fluorescence intensity of cells were detected by flow cytometry. Transfection of different amounts of plasmids containing recombinase genes into cells indicates that cells can produce recombinase at different concentrations. The experiment was repeated three times.
-(A) Fluorescence microscope observation of HEK 293T  undergone different experimental treatments
-(B) The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity.
-(C) The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.
-The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.