Team:NAU-CHINA/Design

Template:2018_NAU-CHINA

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InterLab

Introduction

MOSFET(metal-oxide-semiconductor field-effect transistor) is an essential component in both analog and digital circuits such as analog switches and micro-processors. Inspired by this idea, we built genetic circuit "MOSFETs" in animal T cells which is Monitoring and Operating System Founded on Engineered T cells. We hope our system can serve as a very sensitive bioswitch, which can real-time monitor the extracellular concentration of a certain antigen, and determine whether to activate the expression of a downstream protein according to the preset threshold. As we expect, it should make no response to low concentration, but have quite high sensitivity near the threshold. In order to achieve our goal, we introduced synNotch , TetR-TetO, recombinase and Recombination Directionality Factors (RDF)in our system.

Signal Detection

SynNotch, an engineered transmembrane receptor, bridges intra- and extra-cellular information. Synthetic Notch (SynNotch)[1]consists of three parts, the synthetic extracellular recognition domain (SynECD, e.g.scFv), the core transmembrane domain of wild Notch receptor[2], and the synthetic intracellular transcriptional domain (SynICyi5yD, e.g.SynTF). When the SynECD binds to its targeting surface antigen, induced cleavages take place on the core transmembrane domain of SynNotch, releasing the SynICD. The SynICD would be transported into the nucleus and activate the transcription of its corresponding promoter (Figure 1). SynNotch is an ideal platform for customized antigen sensing behavior. SynNotch provides us an exciting platform because its SynECD and SynICD are both customizable. SynECD can be designed based on currently available scFvs for different tumors .SynICD will trigger customized output after SynECD recognition.

The logic of SynNotch.
The triggering of SynNotch pathway have 4 processes: antigen binding, cleavage,
translocating and promoting transcription. SynNotch is a customizable platform
for cell sensing and response.
Here, we replace the extracellular domain with EGFR- which can recognize cell surface-expressed anti-EGFR and replace the incellular domain with TEV protease which can cleave specific-designed TetR and activate the expression of recombinant enzymes.

Signal Processing

We want to convert the extracellular analog signal into an intracellular digital signal .We use Tet operator (TetO) to achieve this goal. This part contains TetO, TEV protease from the ED of synNotch and Tet repressor (TetR) from 2017 Oxford University iGEM project. The Tet control system is the perfect combination of prokaryotic and eukaryotic gene expression regulation systems. It consists of two parts: the regulatory protein TetR and downstream response elements.

TetR is a repressor from E. coli that blocks downstream expression when it binds to the TetO operon[3]. TEV protease (Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV) [4]. Due to its high sequence specificity it is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.

TetR will bind to the DNA operon and inhibit the production of export proteins. However, TetR has a cleavage site for Tobacco Etch Virus (TEV) protease. When it is cut by TEV, the suppression will be alleviated and the reporter will produce it

Initially，We have thought that we could implement the filtering function by using these elements. When we verificated system by digital-analog, finding that there was a small amount of continuous leakage of the promoter downstream of the (TetO) Tet Operator when there was no extracellular signal stimulation, which was 1% of the normal expression (fig1), but this is still need to improved. To solve the leakage problem, we introduced a recombinase into the system.

Fig1xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

Recombinases , especially a subset called serine integrases and excisionases[5], are enzymes that can flip or excise specific fragments of DNA. Recombinase can directionally catalyze sensitive DNA exchange reactions between targeted short (30–40 nucleotides) sequence sites that are specific to each recombinase. They have been proved to be able to stably modify DNA sequences, which is the biological basis of our MOSFET construct.

Large serine integrases reliably and irreversibly flip or excise unique fragments of DNA . DNA cleavage and re-ligation occur at the central crossover region at a pair of recombinase recognition sites (attB and attP), which allows the sequence to be flipped between recognition sites . After recombination, the original attB and attP sequences become reconstructed sequences - attL and attR. The resulting attL and attR sequences cannot be recognized and bound by integrases alone, so the state after integration is stable.

After digital-analog verification, it was found that replacing the promoter downstream of tetO with the recombinase can simulate different curves. These curves have different thresholds and sudden changes, but they can function as switches that respond quickly. . The feedback was fed back to the experimental team, hoping to switch to different promoters and recombinases to make more diverse switches.

We re-selected the promoters downstream of TetO. And UbC, EF1α, miniCMV were selected and matched with different recombinases, including TP901, Bxb1 and PhiC31, by adjusting the parameters of each part and the strength of the promoter, our whole system has formed nine choices, which can make more threshold choices.

Theoretically, by selecting different promoters and recombinases, we can actually control whether the recombinase can successfully complete its task to reverse its downstream sequence under a certain antigen concentration. In other words, we can preset the threshold antigen concentration according to our practical applications.

Reset

To realize a resettable and more accurate 0/1 switch , we introduce to recombination directionality factor (RDF) in our system. When the external signal disappears or falls below the threshold, our mosfet has a reset function, which is to restore the initial state.

At the second part of our system.We have mentioned recombinases. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL×attR recombination and inhibits attP×attB recombination. Serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL×attR recombination in vivo and in vitro, whereas attP×attB recombination efficiency is reduced. Activation of attL×attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein.

We used RDF corresponding to each recombinase to achieve closure of the device. RDF identifies the attL×attR site and flips it back to the original attP×attB state.

Immediately, we started a new round of mathematical model verification. When the recombinase and RDF existed together, our system would be unstable. While the recombinase brought the system to the 0 state, the RDF would interfere with its behavior and make it into a state. (Fig2). In order to enhance the stability of the system, we have added the RDF-inhibitor part. RDF-inhibitor can act on RDF to make it lose its flipping effect.

Fig2 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

T-cells constitutively express tetR and the promoter is inhibited. When the cells feel the signal in the environment, TEV will bind to tetR, so the recombinase and the inhibitor will be expressed.

When the TEV protein concentration reaches the threshold, the inhibition of the downstream gene by the tetR protein is released by TEV. The recombinase recognizes the attB and attP sites, reverses the sequence between attB and attP, generates new attL and attR sites. The recombinase-RDF and RFP protein can be expressed due to the reversal. The recombinase-RDF loses function by binding with the inhibitor produced upstream. The RFP concentration continues to rise.

When the external signal is weakened and the intracellular TEV protein concentration falls below the threshold, the tetR blocking the expression of recombinase and the inhibitor. Recombinase-RDF recognizes the attL and attR sites ,reverses the sequence between attL and attR, regenerates attB and attP sites and shuts down the expression of RFP protein. To conclude, we can control the quantity of anti-EGFR through T cell automatically.

Reference

1. L. Morsut et al., Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell164, 780--791 (2016).

2. S. J. Bray, Notch signalling in context. Nature Reviews Molecular Cell Biology17, 722--735 (2016).

3.Ramos, J.L., Martínez-Bueno, M., Molina-Henares, A.J., Terán, W., Watanabe, K., Zhang, X., Gallegos, M.T., Brennan, R. and Tobes, R., 2005. The TetR family of transcriptional repressors. Microbiology and Molecular Biology Reviews, 69(2), pp.326-356.

4.Phan, J., Zdanov, A., Evdokimov, A. G., Tropea, J. E., Peters, H. K., Kapust, R. B., … Waugh, D. S. (2002). Structural basis for the substrate specificity of tobacco etch virus protease. Journal of Biological Chemistry, 277(52), 50564–50572. http://doi.org/10.1074/jbc.M207224200

5.Stark WM. 2014. The serine recombinases. Microbiol Spectrum 2(6):MDNA3-0046-2014.