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Synthetic Notch (synNotch)

Synthetic Notch (synNotch) is a novel engineered module receptor that activates the expression of specific target genes when receiving extracellular signals. It consists of three parts, a synthetic extracellular recognition domain (such as single-chain variable fragment (scFv) and Nanobodies), a core transmembrane receptor domain of wild Notch and a synthetic intracellular transcriptional domain. When the extracellular recognition domain binds to its signal molecule, the core transmembrane domain of synNotch releases the intracellular transcriptional domain. It will be transported into the nucleus and activate the transcription of its corresponding promoter. Orthogonal transcription factors such as TetR-VP64 or Gal4-VP64 are used in this process to activate expression of the target gene.

At first, in native Notch, the LNR domains mask the protease cleavage site in the unbound conformation. When the extracellular signal such as antigen exists,the extracellular domain of synNotch such an scFv binds with antigen and the mechanical force will expose this protease site. After a series of reactions, intracellular transcriptional domain will be released and activate the expression of target genes. The extracellular and incellular domain of synNotch can be arbitrarily replaced according to specific experiment requirements.

Here, we replace the extracellular domain with lag 16 which can recognize cell surface-expressed GFP and replace the incellular domain with TEV protease which can cleave specific-designed TetR and activate the expression of recombinant enzymes.

Tet-on system

The TetR DNA binding protein was transformed by the iGEM team of Oxford University in 2017. The Tet control system is a perfect combination of prokaryotic and eukaryotic gene expression regulation system. It consists of two parts: the regulatory protein TetR and its downstream response elements. TetR is a repressor from E. coli which can block its downstream expression when binding to the TetO operon.
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 cleaved by TEV, the repression will be alleviated and the downstream protein can be expressed.

Binding of TetR to its operator site.

TEV protease (Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). Due to its high sequence specificity it is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.

We use TEV protease to remove TetR protein. Therefore, downstream will be turned on.

Recombinase

In a living cell, DNA is the natural medium for storing cell-state information and encoding functions. Recombinases , especially a subset called serine integrases and excisionases, 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, excised, or inserted 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.

Details：In any conservative site-specific （attB&attP）recombination event, there are eight chemical steps: four strand cleavages and four ligations. Cleavage occurs when a nucleophilic amino acid functional group at the recombinase active site attacks the scissile phosphodiester bond of a DNA strand; for the serine recombinases, this is the hydroxyl group of a serine residue. The immediate product of cleavage has a broken DNA strand, with a covalent phosphodiester linkage between one DNA end and the recombinase at the break point. Serine recombinases become linked to the 5′ end of the DNA, leaving a 3′hydroxyl group on the other end at the break. Serine recombinases cleave all four DNA strands in the synaptic complex, creating double-strand breaks at the center of each crossover site. Each half-site thus formed has a recombinase subunit covalently attached to its 5′end, and 2-nt single-stranded protrusions terminated by a 3′-OH group

(Fig. 2). The half-sites are then exchanged and re-ligated, creating recombinants.
FIGURE 2 The serine recombinase strand-exchange mechanism. A synaptic complex of two crossover sites bridged by a recombinase tetramer (yellow ovals) is shown. The four subunitsare spaced out,so that the catalytic steps can be seen clearly. The catalytic serine residues are indicated by S-OH. The scissile phosphodiesters are represented as circled.

Recombination Directionality Factors (RDF)

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. 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.

Details： Mechanism of integrase-mediated recombination. The attP and attB sites are each bound by an integrase dimer (grey ovals), and the two dimers then interact to form a synaptic tetramer (not shown). The DNA strands are then broken and rejoined at the centres of the sites to form attL and attR recombinants. The reverse reaction occurs only in the presence of the RDF (smaller yellow ovals) which binds to integrase and modifies its properties.

Reference
[1]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.
[2]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
[3]Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grünewald, S., Scheek, S., … Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nature Methods, 3(12), 985–993. http://doi.org/10.1038/nmeth967.
[4]Stark WM. 2014. The serine recombinases. Microbiol Spectrum 2(6):MDNA3-0046-2014.
[5]Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C., Colloms, S. D., & Stark, W. M. (2017). Control of serine integrase recombination directionality by fusion with the directionality factor. Nucleic acids research, 45(14), 8635-8645.