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

Engineer 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).

Previous work by Morsut et al. showed that the natural surface antigen CD19 and the non-natural surface antigen EGFP have good orthogonality(7). We applied this and designed two surface antigens, surCD19 and surEGFP. By immunostaining, we were able to clearly detect surCD19 and surEGFP expressed on 293T cells (Fig. 2b). Thus these two surface antigens can serve as ideal dual antigens for our transmembrane binary Boolean logic inputs.

We selected αCD19 (anti-CD19)(6), LaG17 (anti-EGFP with low affinity), LaG16-2 (anti-EGFP with high affinity)(27) as the extracellular domain of SynNotch, mouse Notch1 core regulatory region as the core transmembrane region of SynNotch. Two transcription factors, tTAA (TetR-VP48) and GV2 (Gal4DBD-VP64), which are orthogonal to each other, are used as the intracellular domain of SynNotch (Fig. 2c). We found that the combination of different extracellular domains and intracellular domains has differentiated activation characteristics (Fig. 2d) in transient transfection experiments. In all combinations, αCD19-mN1c-GV2 was not able to be activated efficiently. LaG16-2-mN1c-GV2 and αCD19-mN1c-tTAA have relatively high activation and low background expression. LaG16-2 was able to be efficiently activated only when paired with surEGFP but not surCD19. surCD19 has a similar property (Fig. 2e). The excellent orthogonality between LaG16-2 and αCD19 ensures that we will not undergo signal crosstalk when using dual surface antigens as signal inputs in the future. In the following experiments, we used the LaG16-2-mN1c-GV2 and αCD19-mN1c-tTAA which has good performance, as actual transmembrane signal transduction elements. We used mCherry-tagged LaG16-2-mN1c-GV2 and EGFP-tagged αCD19-mN1c-tTAA to construct a stable cell line with co-expressing of dual SynNtochs for subsequent experiments (Fig. S).

Meanwhile, as we mentioned above, some types of SynNotch have high background expression. This phenomenon aroused our great interest. So we designed a systematic experiment to carry out the fundamental mechanism of SynNotch activation. We designed a systematic experiment to fundamentally study the mechanism of SynNotch activation and hope to optimize it. We specifically covered the details of this section in a SynNotch oriented study in this project.

Figure 2: Engineering SynNotch can receive orthogonal extracellular signals and generate orthogonal intracellular signals.
(a) SynNotch has customizable extracellular and intracellular domains, as well as a core regulatory region. After the extracellular domain of the single-chain antibody recognizes the corresponding surface antigen, the core regulatory region undergoes two cleavages to finally release the intracellular segment of the transcription factor into the nucleus to regulate the downstream circuits. (b) Use surEGFP and surCD19 as ideal antigens. Top left, surEGFP schematic. Green, surEGFP autofluorescence; purple, surEGFP was stained with anti-GFP Rabbit and anti-Rb AF647. Purple shows a clear contour line on the schematic. Bottom left, surCD19 schematic. The extracellular region of surCD19 was labeled with HA tag and stained with anti-HA AF488. Scale bar, 5 μm. (c) Schematic diagram of the SynNotchs we constructed. Different extracellular scFvs and different intracellular transcription factors were used to serve as extracellular or intracellular domains of SynNotch. (d) SynNotch with different extracellular domain and intracellular domains has differentiated expression. SynNotch loaded Receiver cells were stimulated with surAg loaded Sender cells or Mock cells carrying no surAg. the percentage of the cells which highly expressing EGFP (EGFP++%) was analyzed by flow cytometry after 24 hours of co-culture. The error bar indicates SD (n=3). (e) LaG16-2 and αCD19 were able to recognize surEGFP and surCD19 orthogonally. LaG16-2-mN1c-tTAA or αCD19-mN1c-tTAA was activated by surEGFP or surCD19, respectively, and TRE3GV-d2EGFP was used as a downstream reporter. The error bar indicates SD (n=3).

Design three-layer logic processing paradigm for dual transmembrane signals.

The expression of membrane proteins on the cell membrane is limited. For example, cells stably expressing chimeric antigen receptor (CAR) on the membrane are expected to have an expression level in the order of 50,000 molecules/cell(28). It can be compared that in a transient experiment using cationic liposomes, the number of plasmids entering each cell’s nucleus can be as high as 50,000 molecules(29). Therefore, it is critical to design a circuit so that it can accommodate the limited transmembrane signal. Model analysis shows that the use of transcription cascade can effectively amplify the signal.

Based on this, we designed a three-layer transmembrane logic circuit paradigm and limited the transcription units inside the cell to three. The first layer is the "Receptor" layer on the cell membrane, the function of which is to transduce the dual orthogonal antigen signal into the cell by SynNotch to the signal carried by two activating-form transcription factors. The second layer is the "Amplifier" layer within the cell membrane. It contains up to two transcription units. TRE3GV (7xTetO-minCMV) and URE2G (4xUAS-minCMV) are paired activating-form promoters of tTAA and GV2, acting as two channels, receiving signals from two SynNotchs of the "Receptor" layer, respectively. The "Amplifier" layer amplifies the signal from the cell membrane and converts it into a composite signal type carried by engineered zinc finger-based transcription factors. The last layer is the "Combiner" layer within the cell membrane. This layer contains only one transcription unit that integrates the composite signals from the two channels of the "Amplifier" layer and performs the final logical decision output (Fig. 3).

In order to be able to receive dual transmembrane signals and produce 16 complete binary Boolean logic, we designed a set of interactive grammar for the interaction between the "Amplifier" layer and the "Combiner" layer within the cell membrane. This grammar consists mainly of the following elements, a transcription system based on the activating-form, silencing-from or NIMPLY-form promoters (the three are collectively referred to as a synthetic transcription factor-promoter pairs based transcription system); intein-based protein in vivo fusion systems, proteolytic enzyme-based protein in vivo destruction systems (the two are collectively referred to as a protein fusion/destruction-based transcription factor modification system).

Figure 3: The multiple transmembrane signals are processed using a three-layer logic processing paradigm.
On the first layer, the "Receptor" layer, SynNotch receives extracellular signals and then transduces into intracellular signals. The second layer, the "Amplifier" layer, contains two channels, receives signals from the "Receptor" layer, amplifies them and converts them into a variety of signal types based on transcription factors. The third layer, the "Combiner" layer, receives the dual-channel signal from the Amplifier layer and integrates to produce a logical output.

Construct a synthetic transcription factor-promoter pairs based transcription system.

When constructing an artificial gene circuit, it is necessary to consider the interference of the host's endogenous signal. By using a cross-species promoter or synthetic promoter, and ensuring the designed gene circuit to be orthogonal to the host endogenous gene is the key to maintaining system robustness(30). We constructed three mammalian adapted DNA binding domains (DBD) by by using synthetic Zinc finger (SynZF) (31, 32) of ZF21.16, ZF42.10, and ZF43.8. We improved the design paradigm about synthetic transcription factors (SynTF) – synthetic promoter (SynPro) pairs proposed by iGEM 2017 Fudan, constructed three mammalian adapted activating-form transcription factor (aTF) – activating-form promoter (aPro) pairs and three silencing-form transcription factor (sTF) – silencing-form promoter (sPro) pairs to serve as candidate downstream elements of SynNotch. Using a dual-fluorescence dual-plasmid test system, we confirmed that ZF21.16-, ZF42.10-, ZF43.8-VP64 have good activation characteristics (Fig. 4b), ZF21.16-, ZF42.10 -, ZF43.8-KRAB has good inhibition properties (Fig. 4e). At the same time, ZF21.16, ZF42.10, ZF43.8 are orthogonal to each other and to both TetR and Gal4, which are the DBD of selected aTF for SynNotch. By using an identical intermediate transcription factor as a common wiring molecule for the two upstream transcription factors, we can generate an implicit OR gate (Fig. 4f, g) and an implicit NOR gate (Fig. 4h, i).

In addition to traditional activating- and silencing-form promoters, we have also designed a novel NIMPLY-type promoter by adding multiple response elements corresponding to the activating-form transcription factor (aTF REs) and response elements corresponding to the silencing-form transcription factors (sTF REs) at the 5' and 3' ends of minCMV, respectively (Fig. 4j). Thus, the sTF can simultaneously apply (1) transcriptional inhibitors recruited by KRAB can inhibit promoter expression(33); (2) due to RE Located downstream of the promoter, when a sTF binds to the sTF REs, it can cause steric hindrance and enhance the ability of transcriptional inhibition by inhibiting the forward movement of RNA polymerase. This type of promoter exhibits the logical selectivity of NIMPLY type for aTF and sTF. NIMPLY-form promoter can be expressed only in the presence of an aTF and the absence of a sTF (Fig. 4j). 8xZF21.16-minCMV-2xZF43.8 can only be activated in the presence of ZF21.16-VP64 and in the absence of ZF43.8-KRAB, while 8xZF43.8-minCMV-2xZF21.16 can only be activated in the presence of ZF43.8-VP64 and in the absence of ZF21.16-KRAB (Fig. 4k). Through gradient transfection experiments, we also confirmed that the NIMPLY-type promoter exhibits the logic of NIMPLY in the ratio of different aTFs and sTFs (Fig. 4m). In addition, as previously reported cases of aPros(32) and sPros(16) with intensity tunability, NIMPLY-form promoter can also be tuned by using a different number of aRE sites corresponding to the. When the number of sTF REs is fixed and the number of aTF REs is increased, the maximum activation value is on the rise (Fig. 4l).

Figure 4: synthetic transcription factor-promoter pairs based transcription system.
(a), (d), (j). Schematic diagram of the working mechanism of the IDENTITY-, NOT-, NIMPLY-form promoter. An aTF/sTF is constructed by wiring the DNA binding domain (DBD) to a transcriptional activation domain (AD, such as VP64) or a transcriptional silence domain (SD, such as KRAB) via a linker. The IDENTITY-form promoter is just an activating-form promoter. Since it is expressed only in the presence of an aTF, the aPro is expressed as IDENTITY aTF logic. aPro’s structure contains multiple aTF corresponding response elements (aTF REs) inserted in the 5’ terminus of a minimal promoter domain (minPro, such as minimal CMV promoter). The NOT-form promoter is just an sPro. Since it is expressed only in the absence of an sTF, the sPro is expressed as NOT sTF logic. sPro’ structure contains multiple sTF corresponding response elements (sTF REs) inserted in the 3’ terminus of a constitutive promoter (conPro, such as CMV promoter). (b). synthetic aPros can be activated well in the presence of its corresponding aTFs (n = 3, error bar, SD). (c) Orthogonality testing of different DBDs. (e). Synthetic sPro are sufficiently inhibited in the presence of their corresponding sTFs (n = 3, error bar, SD). The dashed line indicates the intensity of expression of the CMV promoter under the same test conditions. Relative to inserting sTF REs in the 3’ terminus of conPro, a 5’-terminus structure can reduce interference with the basal expression of conPro (n = 3, error bar, SD). (f) Schematic diagram of implicit OR. By adding an intermediate layer, signals from aTF1, aTF2 are respectively received using two orthogonally aPros (aPro1, aPro2), then generate the aTF3 as a common wiring molecule. (g) implicit tTAA OR GV2 gate. Using tTAA, GV2 as the input signals. The TRE3GV, URE2G promoters receive signals from tTAA and GV2, respectively. ZF21.16-VP64 was used as aZF3, and its downstream 8xZF21.16-minCMV promoter controls the expression of d2EGFP (n = 3, error bar, SD). (h) Schematic diagram of the implicit NOR gate. Unlike implicit OR gate, here we use a sTF as a common wiring molecule. (i) implicit tTAA NOR GV2 gate. sTF1 is ZF21.16-KRAB, and its downstream 8xZF21.16-CMV controls d2EGFP expression (n=3, error bar, SD). (k). The NIMPLY-form promoter is highly expressed in the presence of aTF and in the absence of sTF. When aTF and sTF coexist, sTF plays a major role. (I) The maximum activation intensity of the NIMPLY-form can be tuned by changing the number of repeats of the aTF REs. (m). The NIMPLY-form promoter shows NIMPLY logic selectivity at different aTF, sTF levels.

Construct a protein fusion/destruction-based transcription factor modification system

To build more complex logic systems, we used intein and proteolytic enzyme technologies. Cfa is a highly efficient fast split intein(34). Cfa’s residue tolerance of the C-extein proximal sequence was improved by directed mutagenesis of the catalytically related key loop(35). We split ZF21.16 in the first cysteine(31) of the outer region of the ZF structure, resulting in the N-terminal ZF21.16 (ZF21.16'N) and the C-terminal ZF21.16 (ZF21.16'C) . We co-transfected VP64-ZF21.16'N-Cfa'N and Cfa'C-ZF21.16'C into the cell. Comparing with the cell only transfected the VP64-ZF21.16'N-Cfa'N or Cfa’C-ZF21.16’C, the co-transfected cells showed a relatively higher activation level of 8xZF21.16-minCMV (Fig. 5b). Similar experiments were performed using KRAB-ZF21.16'N-Cfa'N and Cfa'C-ZF21.16'C, and expression of the 8xZF21.16-CMV promoter in the co-transfected group was inhibited (Fig. 5d). Combining use of the aZF’N and aZF’C can generate the AND gate (Fig. 5a), while the co-effect of sZF’N and sZF’C results the NAND gate (Fig. 5c).

We next tried to use proteolytic techniques to build more complex logic gates. Due to the stringent sequence specificity of Tobacco etch virus protease (TEVp), it is widely used in the field of synthetic biology(20, 23). We replaced the amino acid sequence of the ZF21.16-KRAB linker region by using the high-affinity TEV cleavage sequence (TCS), and added a certain of Gly or Ser at both ends of the TCS to provide flexibility and then constructed destroyable sTF (dsTF). dKRAB-ZF21.16 has similar inhibition ability to 8xZF21.16-CMV as ZF21.16-KRAB (Fig S). In the experimental group which co-transfected with dsTF and TEVp, the expression of inhibited 8xZF21.16-CMV was partially restored (Fig. 5f). Therefore, the IMPLY logic can be constructed (Fig. 5e).

Figure 5: Protein fusion/destruction-based transcription factor modification system can be used to construct more complex logic.
(a). Construct an AND gate by using split aTF and intein. aZF'N is a fusion of AD (VP64) and the N-terminus of DBD (DBD'N) with the N-terminus of Cfa (Cfa'N). While aZF’N meeting with aZF’C (consisted of DBD’C and Cfa’C), rapid and seamless assembly is performed, which in turn generates the active aTF to induce its corresponding aPro. (b). Construct an AND gate with VP64-ZF21.16'N and ZF21.16'C. When VP64-ZF21.16'N and ZF21.16'C are present simultaneously, the expression of downstream 8xZF21.16-minCMV relatively rises. (n = 3 technical repeats, error bar, SD). (c) Constructing an NAND gate by using split sTF and intein. Use KRAB as the SD. When both sZF'N and sZF'C are present, active sTF is produced to inhibit its corresponding sPro. (d) Constructing NAND gate with KRAB-ZF21.16'N and ZF21.16'C. The downstream sPro is 8xZF21.16-CMV (n = 3 technical repetitions, error bar, SD). (e) IMPLY gate can be achieved by using dsTF and TEVp. The linker of dsTF between SD and DBD contains a TEVp high affinity sequence. When TEVp is present, the linker of dsZF is destroyed, and KRAB and DBD are separated. At this time, even if the DBD is able to bind to the sTF REs on the sPro, it is unable to suppress the expression of sPro due to the lack of SD. (d) Constructing NAND gate with KRAB-ZF21.16'N and ZF21.16'C. When TEVp and dKRAB-ZF21.16 are simultaneously present, TEVp disrupts dKRAB-ZF21.16, resulting in a certain degree of recovery of downstream 8xZF21.16-CMV expression (n = 3 technical replicates, error bar, SD).

So far, we have tested all the components needed to build intracellular logic gates, and they all have the ability to work. At the same time, we completed most of the construction of 16 intracellular logic gates by using the synthetic transcription factor-promoter pairs based transcription system and the protein fusion/destruction-based transcription factor modification system, which we summarize below (FigS). Our next goal is to integrate these intracellular elements with “Receptor” layer to construct transmembrane binary Boolean logic.

transmembrane binary Boolean logic

Any of the 16 transmembrane binary Boolean logics can be constructed by integrating the "Receptor" layer in which SynNotch is located with the intracellular logic elements we constructed above (Fig). Here, we use the OR gate as a proof of concept for a three-layer paradigm-based transmembrane binary Boolean logic. In our test, we used the previously constructed bistable cells as the cell chassis of the Receiver cell, on which the elements of the "Amplifier" and "Combiner" layers corresponding to the OR gate were transiently transfected. LifeAct-EGFP-labeled 293T-surEGFP and H2B-EGFP-labeled 293T-surCD19 were used as Sender Cell. In this way, we can clearly distinguish the type of antigen carried by the sender cells according to the position of EGFP under the microscope. Microscopy showed that both surEGFP (Fig. 7a) and surCD19 (Fig. 7b) were able to activate our Receiver cells, respectively. This shows that both the surEGFP and surCD19 signals can be received by the SynNotch paired with them and passed to the final "Combiner" layer via the "Amplifier" layer. Therefore, we have conceptually demonstrated the feasibility of the transmembrane binary Boolean logic based on the three-layer paradigm.

Figure 6: Using the three-layer paradigm to build a transmembrane OR gate.
(a) A schematic diagram of the OR gate Receiver cell. (b). OR-Receiver can be activated by surEGFP to generate a signal. Bottom right, pattern mode diagram. S, Sender cell with surEGFP. A, activated Receiver cells. I, inactivated Receiver cells. Receiver cells that are in direct contact with sender cells are activated, and Receiver cells that aren’t contacted by sender cells cannot be activated. U, Receiver cell that failed to transfect the OR gate intracellular element. (d) The Receiver cell equipped with an OR gate can be activated by the surCD19 to generate a signal. The Z-Projection projection was used to illustrate the dynamical trajectory of cell movement for 30 hours (Movie). Scale bar, 20 μm. We firstly transfected the dual stable Receiver Cell with 293T-LaG16-2-mN1c-GV2 and αCD19-mN1c-tTAA on two glass Petri dishes that have been Coat by fibronectin. at 0h. the required elements of OR gate’s "Amplifier" and "Combiner" layers were transiently transfected into two culture dishes in duplicate. After 8 hours of transfection, the two sender cells were plated onto Receiver cells separately. For the experimental group using surEGFP Sender cell, we detected the SynNotch activation at 54 hours after transfection. For the experimental group activated with surCD19 Sender cell, the continuous live motion was taken for 30 h at 24 h after transfection using a live cell workstation. The picture shown is a representative picture of all fields of view taken. We have removed the weak background green fluorescence of SynNotch stable cells and surEGFP cells in the picture.

Discussion

Single cell-based logical operation group

In the ENABLE system, it is theoretically possible to construct 16 single-cell logic calculators with different dual signal processing logic. These cell-based logic calculators have customizable input and output units that inspire us to define cell functions at a new level. We named this new concept "Cellbrick". Cellbrick is similar to Biobrick, a well-known modular concept at gene level for synthetic biology, but is even more different in that it is actually a standardized biological device based on cell levels. In the Cellbrick system, each cell can be explicitly defined as a microprocessing unit with specific inputs and outputs. By using different combinations of input and output, Cellbrick is able to perform specific tasks in a coordinated manner in terms of processes or results (Fig.).

In particular, Cellbricks can be “wired” by standardizing orthogonal surface antigens, allowing cells and cells to be connected in a gear-like or brick-like manner. Different Cellbricks form an interaction system with upper and lower levels. By optimizing the performance of each Cellbrick, the overall interaction system can perform a wider range of functions at a higher level. Previous logic gate systems often have mismatches in the input signal-output signal class (For example, we could design circuits that enable cells to logically process complex small molecule signals. However, because the output port product is often a protein rather than a homogeneous small molecule. That will result in an irreversible signal transmission chain(10).) or the type of input signal cannot spontaneously cross the cell membrane into the cell in a natural way (For example, input the signal by transient transfection(16).). These logic gate designs can only choose to perform centralized computing(10, 15, 16, 18, 19) (in simple terms, the process of logical operations is limited to one cell) or distributed computing(13, 14, 17) (in simple terms, that is, the logical operation can be performed by any of a plurality of cells or cell groups.). However, by using a library of Cellbricks which are spatially wired by antigen, we can allow cells to perform intercellular iterative distributed computing systems in a cell-cell contact-dependent manner. Moreover, this kind of distributed computing also has the characteristics of centralized computing. For example, by correlating Cellbricks with different logical operations (the internal operations of each cell actually run in a centralized computing way), or the Cellbricks equipped with the logic gate with logical complete such as NOR, we can construct a very complex logic processing system in theory (Fig).

Building Cell Legion with Cellbricks

In today's cellular immunotherapy, people tend to focus on the use of tools such as CAR to customize a straightforward immune response (recognition → killing). In contrast, the natural immune system achieves extraordinary regulation through a highly complex and networked interaction between multi-cells. Just by expanding the cell killing function of the TCR pathway, the CAR system shows such an exciting clinical application prospect(40). If there is a method that allows people to fully invoke the specialized mechanisms in the natural immune system, would it allow cell immunotherapy to enter a new era? For example, in cellular immunity, the interaction of helper T cells and effector T cells plays a key role(41). But as far as we know, there is still no system that attempts to simulate this natural mechanism and use multiple cells for interactive design. We imagine that through the Cellbrick system, people can think and customize cell-cell interaction logic in a new dimension. In the future, people may be able to cope with the challenges of various diseases by using a more ingenious design and building a Cell Legion with different functions based on a comprehensive consideration of the challenges faced by different diseases(42).

Future work

Further exploration of SynNotch design principles

In order to achieve complete transmembrane binary Boolean logic, in addition to using our well-designed three-layer transmembrane logic paradigm. In our initial trials, we attempted to directly use different synthetic activating-, silencing-form transcription factors to serve as the intracellular domains of SynNotch, attempting to construct a logic gate system just by the transcriptional network. But disappointingly, these novel SynNotchs equipped with synthetic zinc finger-based transcription factor were not able to be efficiently activated (Fig. S). We hypothesize that this may be due to two reasons: 1. We use a three-finger synthetic zinc finger protein to construct a SynTF that recognizes an RE sequence specific to about 9 bp, which may limit its extent to bind the RE with high affinity. Therefore, it may be necessary to reach a certain working concentration to drive the function. Since the expression of SynNotch on the cell membrane is limited, when the SurAg activates SynNotch, only limited intracellular domains can be cleaved to produce an effect. These may lead to the disability of SynZF-TF. Therefore, we design the excellent three-layer transmembrane logic paradigm to solve this problem. At the same time, we are also using the newly designed SynTFs based on six-finger zinc finger protein to construct a novel SynNotch. These novel synthetic six-finger zinc finger proteins recognize response elements up to about 18 bp and theoretically have superior affinities compared to the three-finger one. 2. As mentioned in the initial research by Lim, although the intracellular domain of SynNotch has excellent programmability, in practice, not all the combination of the extracellular and intracellular domains can be grafted to SynNotch with function(6). In our fundamental research on SynNotch, we found that its S3 cleavage site has crucial biological functions. We believe that it is possible that the configuration of SynZF interferes with the proximity of γ-secretase and that SynNotch cannot be efficiently cleaved. In this regard, as we mentioned in the work of the SynNotch Optimization project, we are trying to design a more versatile linker for the joints at both ends of the SynNotch core region to extend the broad adaptability of the SynNotch design.

Further optimization of the protein fusion/destruction-based transcription factor modification system

In our experiments, it was found that transcription factor modification systems based on protein fusion/destruction are still insufficient efficiency. Although Cfa is the fastest assembled and robust intein known to us, it only exhibits limited assembly efficiency in our application systems (such as AND and NAND gate). At present, we believe that the following two possibilities may limit its efficiency: 1. We split the C2H2-type ZF21.16 at the first Cys site on the first β-fold of the second zinc finger motif and then construct the ZF21.16’N-Cfa’N and Cfa’C-ZF21.16’C. Although the Cys split site, in theory, will not cause scarring of ZF21.16 after assembly of the intein, it is still possible to interfere with the natural conformation of ZF21.16 to some extent after incorporation of the intein. 2. As previously reported, Cfa constructed based on the DnaE type intein groups still retains the preference for a bulky hydrophobic residue (e.g., Phe) at the +2 position of C-extein(34). In subsequent work, we are ready to try other cleavage sites and follow the stricter Cfa C-extein proximal amino acid preference rules for design or use a more appropriate linker to design the Cfa-split SynTF.

Construction of complete transmembrane logic gates

Limited to the time factor, in this year's work, we only use the OR gate as a proof of concept for the transmembrane logic gate. But as we have shown, we have completed most of the intracellular logic gate construction and characterized its performance. We believe that the construction of complete transmembrane logic gates will be just around the corner in the near future.