Introduction

Sensing and integrating various transmembrane signals is a key aspect of cellular decision making. For example, activation of CD8+ cells requires co-activation of TCR and CD28 molecules, meanwhile, this activation can be inhibited by the PD-1 pathway(1). By abstracting this biological process, we can get: the activation of CD8+ cell = activated TCR AND (activated CD28 NIMPLY activated PD-1) (Fig. 1a). Programming cells with predictable complex transmembrane signal inputs – customized intracellular signal outputs logic relationships are significant for expanding the widespread applications of mammalian cells, such as cellular immunotherapy(2-5), tissue patterning(6, 7) (Fig. 1b).

Figure 1: The logical integration of complex transmembrane signals has crucial biological implications.
(a) Immune cells recognize target cells by integration of multiple transmembrane logic signals. Immune cells need to be co-stimulated by TCR and CD28 to be activated. Tumor cells can induce PD-1 activation of immune cells by expressing PD-L1 molecules. PD-1 can, in turn, interrupt the progression of the immune response by terminating the co-stimulatory signal of CD28. Thus, tumor cells cannot be recognized by immune cells and cause immune escape. (b) Engineering the cells to perform logical operations on multiple transmembrane signals to produce a variety of customizable outputs could enable cells to gain novel application potential.

The transmembrane signal transduction is obstructed by the cell membrane, a unique feature that makes designing multiple transmembrane signal sensing and integrating systems a huge challenge. As we have seen, synthetic biologists have achieved great success in the integration of non-transmembrane signals. Although researchers already built partially(8-12) or completely(13-19) binary logic gates in both prokaryotes(8, 9, 11, 12, 15, 17, 18) and eukaryotes(10, 13, 14, 16, 19), all of these mentioned design limited by they can only integrate signals that are confined within the cell membrane(11, 12, 16, 19), or are the small molecules which could freely penetrate the cell membrane(8-10, 13-15, 17, 18). Therefore, developing a complete system that enables it to sense and integrate complex transmembrane signals is a current core challenge for synthetic biology.

We designed the ENABLE (Engineered, Across-membrane, Binary Logic in Eukaryotes) system and achieved the first complete transmembrane binary Boolean logic in mammals. The three-layer modular design (Receptor, Amplifier, and Combiner) of the ENABLE system gives the system great expandability, which not only promising a host of application potentials but also provides a design paradigm for the future transmembrane logic decision system. At the same time, the ENABLE system is not only capable of running single-cell-based centralized logic calculations but also enables distributed logic calculations based on single-cell-single-cell contacts through spatial wiring by the surface antigens. These characteristics give the ENABLE system the possibility to perform the sophisticated cellular computation.

Result

Engineering SynNotch enables it to receive extracellular signals and generate orthogonal intracellular signals

In order to be able to implement a custom multiplexed transmembrane signal input/output relationship, the first condition is that engineering modular receptor to enable it to recognize extracellular signals and transduce them into customized intracellular signals. To this end, a variety of techniques have been developed, such as Tango(20), CAR(21), GEMS(22), MESA(23-25), SynNotch(4-7), etc. High programmability of the extracellular and intracellular domains of SynNotch, as well as adaptation to contact-dependent signaling, fully meets our needs. Thus we ultimately apply SynNtoch technology as a receiving port for extracellular signals of transmembrane logic gates.

Synthetic Notch (SynNotch) has a minimal core regulatory region of Notch receptor(6). By wiring it to a chimeric extracellular domain (such as a single-chain antibody) and a chimeric intracellular domain (such as a transcription factor), SynNotch can recognize customized surface ligand signals and produces customized intracellular outputs. In the mechanical force activation model(26), when SynNotch recognizes a homologous ligand on an adjacent cell, its minimal core regulatory region will undergo a series of cleavage and eventually release the intracellular domain into the nucleus, and drives the expression of the user-specified downstream circuits (Fig. 2a).

Strengths of TALE-NOR gates

• They optimized their NOR gates by placing the operators upstream from the promoter as it produces minimal variability in reporter expression and retains efficient repression.
• They use the SAME type of NOR gates for all their circuit functions and allows for the construction of complex circuits with precision and interoperability.
• The implementation of TALE-based cellular circuits could be easily extended to other cellular chassis, such as yeast or bacteria.
• TALEs can accommodate only one or two mismatches out of the 18-nucleotide-recognition site, supporting the potential for large orthogonality.

Integrate TALE-NOR based logic gates with ENABLE

Using NAND gate as an example

Figure legend in the original study: Implementation of the two-input Boolean logic NAND function constructed from combinations of designed TALE repressor-based NOR gates.
The logic connectivity of biological NOR gates and experimental results from the firefly luciferase and confocal microscopy are shown, with the truth table below the luciferase reporter results. Their NAND gate, constructed from two NOR gates, will produce an output if at least one of the two signals is off. Only those TALE binding sites that were functionally relevant for the circuit are illustrated. The appropriate combination of processing logic and input plasmids were introduced into HEK293T cells for each combination of input values. Values are the mean of n = 4 ± s.d. For all functions, the statistical significance of the separation between the high and low states is significant at the level ***P < 0.001. Microscopic images are representative of five separate observations. Scale bars, 125 μm. nRLU, normalized relative light units.

ENABLE^TALE-NOR

ENABLE^TALE-NOR will use our Receptors to pass the signal across the membrane and will use our transcriptional modules to amplify the production of TALE-NOR transcription factors. Different combinations of TALE-NOR will behave as our Combinator to execute binary logic functions.

HOW TO IMPLANT ENABLE^T2

In summary

• The authors used RNA-binding proteins and their RNA target units to create a set of synthetic transcription-translation control devices that can be rewired in a plug-and-play manner.
• The transcription factors are triggered in the sense that they are activated only when they interact with specific inputs.
• They use RNA-binding proteins that inhibit the translation of transcripts containing specific RNA target units.
• They used a half-subtractor and half-adder to perform fundamental arithmetic operations (addition and subtraction) of two bits. Their half-subtractor was made by a combination of the XOR gate and the NIMPLY gate. Their half-adder was made by a combination of the XOR gate and the AND gate. For more details, please refer to Figure 4.
• They have achieved two-input-two-output integration.

Side-by-side comparison

Strengths of transcription-translation control devices

• These transcription-translation control devices are rational and has a predictable plug-and-play characteristic. Individual components can be readily rewired to perform computing activities, such as NOT, NAND, and NIMPLY.
• Their single-cell biocomputers are scalable to tissue structures and are straightforward to wire with host metabolism to have therapeutic impact.

Integrate T2 into ENABLE

Using NIMPLY gate as an example

Figure legend in the original study: Design synthetic NIMPLY gates in human cells.
(a) A ANDNOT B logic gate. By combining the two input signals erythromycin and phloretin in accordance with the truth table, transfected HEK-293 cells are programmed to produce d2EYFP exclusively in the presence of erythromycin and not phloretin as shown by fluorescence microscopy and FACS analysis. b.t., below the threshold of 104 fluorescence units. Error bars represent s.d.; n = 3.

ENABLE transcription-translation control devices

ENABLE^{transcription-translation} (ENABLE^T2) will use our Receptors to pass the signal across the membrane and will use our transcriptional module to amplify the production of trigger-controlled transcription factors, including both RNA-binding proteins and their RNA target units. Different combinations of trigger-controlled transcription factors will behave as our combinator to execute binary logic functions.