Team:NAU-CHINA/Demonstrate

Template:2018_NAU-CHINA

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Demonstrate

Overview

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.

1)Verify the function of each part.

2)Combine the parts into two large modules of upstream and downstream circuits to verify the function.

3)Assemble the upstream and downstream circuits to verify the function of the whole system.

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.

Fig.1. Verification of the localization of Lag16-synnotch-TEV on cell membrane
Transfect HEK 293T with plasmid containing Lag16-synNotch-TEV. Mix cells with GFP, and incubate for 30 minutes. PBS wash away free GFP.
(a) (a) Fluorescence microscope observation of the cells cross-linked with GFP. The results showed that synNotch can be located to the membrane.
(b) Blank control (without transfection).
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.

Fig.3. Inhibition of tetR on promoter with tetO sequence
(a) Fluorescence microscope observation of HEK 293T only transfect with plasmids containing promoters with tetO sequence.
(b) Fluorescence microscope observation of HEK 293T transfect with plasmids containing promoters with tetO sequence and tetR.

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 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

Figure4. Function verification of recombinases in HEK 293T cell

Fluorescence microscope observation of HEK 293 T transfect with plasmids containing the recombinase recognition sites and corresponding recombinase 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 recombinases 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

Fig.5. Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells
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) 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.(b) The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump between low concentration and high concentration of TP901 recombinase. Similar results were obtained in all three repetitions.

The results of image a 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 in fluorescence intensity. The results of the right image b show that TP 901 recombinase has a threshold property. So, the proportion of fluorescent cells have a jump discontinuity. Although the data of the three repetitions are all similar results, this jump discontinuity is only about 4 - 5 %. If we need to prove the existence of the threshold, further experiments are still needed.

Function verification of RDF in HEK 293T cell

Fig.6. Function verification of the reversal ability of recombinase-RDF 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. Function 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. Then the TEV gene was transferred into the aforementioned stably transferred cells. Through fluorescence microscopy and flow cytometry, we could 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 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.Further verification of threshold characteristics of recombinases

In the experiments we have done, although we have got a jump discontinuity, it is not obvious due to the problem of plasmids randomly transferring into the cell. Moreover, more concentration points need to be selected. 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 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.

5.Upgrade our system

The above mentioned is only a condensed version of our ultimate system which include RDF and inhabitor. We hope to upgrade the condensed version to the final version, which also requires eukaryotic function verification of RDF and the search for appropriate inhabitor. 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).

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