Upstream circuit

The upstream circuit mainly designs a signal path to enable cells to receive specific external signals and activate the downstream core path to realize the threshold function. Therefore, the upstream circuit can be replaced according to the situation. Here we provide an upstream circuit design as a reference and other researchers can design an upstream path according to their own needs.

Customizing the signal path of cells in response to external signals

There is a wide type of extracellular signal. Cells receive extracellular signals and will respond to the signal molecules accordingly. We hope to customize a receptor so that it can recognize the signal molecules and regulate downstream gene expression. We choose synNotch as a kind of ideal receptor.

Similar to some signal moleculars, take Epidermal Growth Factor Receptor as example which is our realistic system, the GFP is also protein but more stable and no impact on the system.

And as for visibility and operability, cell surface-expressed GFP as model of extracellular signal molecular is a better choice. So we want to replace the excellular domain of synNotch with Lag16 which is 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 (Part名) and synNotch (Part名) from iGEM 2018 Fudan team. But the intracellular domain of synNotch from is tTA which a kind of activation factor. Since synNotch was applied to the transformation of cells, the intracellular domain has been replaced by transcription activators such as GAL - VP64 and tTA(引用文献）. However, promoters are not completely non-expressed until they are activated and they often have background expression（引用文献）. Moreover, we hope to make some changes to the intracellular domain of syNotch, try to replace the intracellular domain with non-transcriptional activator substances, and broaden the selection and application of synNotch intracellular domain.

We found that the previous team iGEM 2017 Oxford modified tetR by replacing tetR DNA binding domain and regulatory core domain with TEV enzyme cleavage site, so that tetR would 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 IGEM 2017 Oxford's idea, we replaced synNotch's intracellular domain with TEV, and repeated verification of synNotch's function to explore whether replacing intracellular domain with TEV of non-traditional transcription activator will affect synNotch.

The above two experiments show that the modified Synnotch can be positioned on the surface of cell membrane normally and can release intracellular domain after receiving external signals. The replacement of intracellular domain with hydrolase has no effect on the function of synNotch

Eukaryotic Verification of TEV Activation Transcription System Based on Modified tetR

As mentioned earlier, inducible promoters that use transcription activators that cannot inhibit transcription often have some leakage due to background expression.

However, our system hopes to realize the absolute functions of 0 and 1, and the background expression is what we don't want to see. Therefore, we need to find a transcription activation system with very low background expression.

Coincidentally, the previous team IGEM 2017 Oxford is also making a 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 convincing. Therefore, we decided to try to verify their system with eukaryotic cells.

The result shows that tetR can effectively repress the expression of green fluorescent protein in the promoter with tetO sequence.Unfortunately, due to time constraints, we have only partially verified the system. We have carried out the verification of tetR inhibition, and the verification of TEV elimination of TETR inhibition needs to be carried out later.

Downstream circuit

The downstream pathway is the core circuit for us to realize the threshold function. According to Lu Guanda's (引用文献）literature, 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 recombinase concentration reaches a certain threshold can the recombinases work normally.

According to this document, we designed our pathway in eukaryotic cells, expecting to realize threshold switching in eukaryotic cells. For this reason, we tested the recombination enzyme inversion function and threshold characteristics of the combination of three different recombinases ( BXBI 1, TP 901, φ C31 ) and three promoters with different intensities ( Mini CMV, EF1 - α, CMV - enhancer ) in eukaryotic cells.

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

Fluorescence microscope observation of HEK 293 T transfected with plasmids containing the recombinase recognition sites and corresponding recombinase gene .Transfection of different numbers of plasmids containing recombinase genes into cells indicates that cells can produce recombinase at different concentrations.

On th 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 showed that the recombinases can recognise the sites and reverse the sequence between sites in HEK 293T.

Functional Verification of Reversal Efficiency and Threshold Characteristics of different recombinase in HEK 293 T Cells

The results of the right image show that the reverse efficiency of Bxb1 recombinase is higher than TP 901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference fluorescence intensity. The results of the right image show that TP 901 recombinase has a threshold property TP 901 recombinase has a threshold property. So the proportion of fluorescent cells will jump. Although the data of the three repetitions are all similar results, this jump is only about 4 - 5 %.

If we need to prove the existence of the threshold strongly, further experiments are still needed.

Conclusion

We verified most of the part and upstream and downstream paths step by step. We verified the function of synNotch, the inhibition of tetR after modification, the recombination function and threshold characteristics of some recombinase and promoter combinations. However, due to time constraints, we were unable to complete verification of TEV and some recombinase and promoter combinations. Moreover, the combination of upstream and downstream channels 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.

1.Functional Verification of TEV suppressing tetR Inhibition

We plan to stably transfer the modified TetR gene and the promoter with TetO sequence into the cell and construct a stably transferred cell line. The TEV gene was then transferred into stably transferred cells. Through fluorescence microscopy and flow cytometry to observe the fluorescence intensity of cells and the proportion of fluorescent cells to determine whether TEV can relieve the inhibition of tetR.

2.Verification of the combination 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.Further verification of threshold characteristics of recombinases

In the experiments we have done, although we have got a sudden jump, this sudden jump is not obvious due to the problem of random plasmid transfer into the cell. Moreover, fewer concentration points were selected. So persuasion needs to be further strengthened. After that, we will strongly verify the threshold characteristics of recombinase in eukaryotic cells by constructing stable cell lines, increasing the gradient and detecting the concentration of recombinase by Western and qPCR.

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

We plan to finally build our Parts on two plasmids. Stable cell lines with complete functions were constructed through Puro and BSD screening, and their concentration threshold functions were verified by using agarose beads with different amounts of GFP adsorbed. And try to apply it to real life.

5.Upgrade our system

The above mentioned is only a condensed version of our ultimate system which still include RDF and INHABITOR. We hope to upgrade the condensed version to the final version, which also requires eukaryotic verification of RDF's functions and the search for appropriate RDF. We look forward to the day when our final version will appear.