Team:NAU-CHINA/testh

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 gene. 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).