Harnessing Naturally Occurring Bistable Switches for Their Application in Synthetic Biology

Abstract

Bistability is a fundamental phenomenon in nature. In biochemical systems, it creates digital, switch-like outputs from the constituent chemical concentrations and activities, and it is often associated with hysteresis in such systems. Here, we first introduce the regulation of bistable switches at different levels in natural life systems, then explain the current pioneering applications of bistable switches in synthetic biology, and finally introduce some design and tuning methodologies and principles that may be helpful for the future application of bistable switches in synthetic biology.

1. Introduction

Living systems maintain internal stability owing to their natural tendency to resist external forces of change. The general idea of self-regulation was put forward by Clause Bernard, and the word homeostasis was coined by Walter Bradford Cannon [1,2]. Homeostasis is thus a vital biological process that is ongoing in all living systems, including intracellular signaling pathways, single cells, cell–cell communications, tissues, organs, whole organisms, and populations (e.g., prokaryotic communities). To advance life processes or to adapt to the environment in a timely manner, a living system often needs to switch flexibly between steady states to achieve optimal homeostasis, for which bistability has evolved [3]. Through hysteresis, a bistable switch prevents constant switching between steady states, thereby sustaining the current steady state and producing a memory of a transient stimulus. Once a change in biological parameters crosses a threshold, the system abruptly switches between different steady states, thereby providing timely switching between alternative modes of regulation. Therefore, bistability has wide and profound manifestations in natural life systems. The discovery of various bistable switches in natural life has also brought great inspiration to the field of synthetic biology. Researchers have explored bistable switches from both methodology and application and have made some creative breakthroughs. This article first introduces the regulation of bistable switches at different levels in natural life systems, then explains the current pioneering applications of bistable switches in synthetic biology, and finally introduces some design and tuning methodologies and principles that may be helpful for the future application of bistable switches in synthetic biology.

2. Naturally Occurring Bistable Switches

There are several regulatory switches found in natural biological systems, including monostable switches, multistable switches, ultrastable switches, oscillatory switches, excitable switches, and quorum-sensing switches [4]. As the most dominant kind of multistability, bistability is widely found in natural life systems ranging from microscopic to macroscopic levels (Figure 1). Bistability remains an intriguing natural phenomenon until its underlying mechanism is gradually revealed. From the activities of non-cellular structures to the regulation of the inside and outside of cells and to the regulation of individual systems of multicellular organisms, bistable switches occur in both classic genetic transcription regulation and cellular signaling pathways and also cleverly couple different types of cellular events, thereby achieving a wide range of biological functions.

2.1. In Simple Organisms

In the study of non-cellular viral systems, the lysis–lysogeny decision of bacteriophage lambda (λ) was discovered early on to be regulated by a bistable switch consisting of a pair of gene repressors: CI and Cro (Figure 1a). While CI promotes phages to enter the lysogenic state, Cro promotes phage to enter the lytic state. A previous review has provided us with comprehensive knowledge about the in vivo genetic network regulating this bistable switch, in which CI and Cro do not simply regulate each other directly but contain more mutually regulating regulatory genes, complexes, and antisense RNAs [5]. Another recent review highlighted mathematical modeling of the in vivo system to explore this bistable decision [6]. Interestingly, while mathematical modeling demonstrated better performance of the in vivo system, which includes more essential regulating factors in the network [7], a later in vitro experiment found that CI and Cro can directly form a bistable switch [8]. By comparing the differences in the lysis–lysogeny bistability between in vitro and in vivo settings based on mathematical modeling of the in vivo molecular network, researchers may gain new insights into the regulatory mechanisms of bistable switches.

In the study of prokaryotes, researchers have long noticed that E. coli switches lactose metabolism when switching between glucose and lactose feeding cultures [9]. Subsequently, the lac operon was proposed and continuously studied as a lactose-metabolic genetic switch. The lac operon has been well described in a review article, particularly its components and mathematical models [10]. When a non-metabolizable lactose analog thio-methylgalactoside (TMG) is used as an inducer in the lac operon experiment, a clear bistable switch can be observed (Figure 1b), which can be explained by the corresponding mathematical model [11]. However, it is difficult to observe the bistable switch when only natural lactose is used as the inducer [10]. A mathematical modeling study showed that the extracellular glucose vs. lactose concentration parameters for establishing bistability would change when TMG and natural lactose are used simultaneously as inducers. It was also inferred that extremely high concentrations of lactose would be required to observe a bistable switch using only natural lactose inducers [12]. This aspect requires more experiments and models to verify and explain.

2.2. In Eukaryotic Cells

In the study of eukaryotic cells, complex bistable switches have been revealed in the intracellular signaling pathways. More than 20 years ago, a review summarized and hypothesized the cellular bistable signaling that had been partially revealed at that time, including part of the cell cycle transition, the mitogen-activated protein kinase (MAPK) cascade, and the c-Jun N-terminal kinase (JNK) cascade [13]. Subsequently, more cellular bistable signaling cascades were discovered.

Bistability is recurring in, and is thus intensively studied in the context of, cell-cycle transition. Cell cycle activators usually inactivate inhibitors through multistep modifications, whereas hypo-modified inhibitors antagonize the enzymatic activity of activators by binding to them [14]. Three specific checkpoints, including G1/S transition in budding yeast, prometaphase-to-anaphase transition, and mitotic exit in human cells, are regulated, respectively, by a bistable switch consisting of different pairs of activators and inhibitors, and they are characterized, respectively, by a regulatory inhibitor-regenerating enzyme that is inhibited by the cell-cycle activator (Figure 1c) [15,16]. Periodic bindings of cyclin-dependent kinases (CDKs) to cyclins in different phases of the cell cycle constitute different bistable switches and act as cell cycle latches in eukaryotes [17]. As described in a review [18], a bistable switch consisting of retinoblastoma protein (Rb) and E2F family transcription factors acts as, besides a switch, an integrator and actuator during cell cycle progression. Rb integrates upstream signals and transcriptionally represses E2Fs, while E2Fs induce their own expression and activate CDK2 to reciprocally inhibit Rb. For cell cycle transitions, bistability has been demonstrated to have extremely high robustness and accuracy in temporal and spatial control [19]. More sophisticated cell regulation can be achieved by coupling cell cycle transitions with other signaling pathways. In Xenopus oocyte maturation, for example, the positive-feedback-based bistable module consisting of p42 MAPK and cell-division cycle protein kinase Cdc2 enables the system to realize irreversibility of maturation with transient inductive stimulus of oestradiol. When this positive feedback loop is blocked, a transient stimulus only generates a transient response, which is unable to sustain maturation. Because p42 MAPK and Cdc2 are positive feedback themselves, the system is a further positive feedback between two positive feedbacks [20]. In lymphoid and myeloid differentiation, the accumulation of transcription factor PU.1 and the cell cycle lengthening constitute a positive-feedback-based bistable switch to control cell fates. Decreased PU.1 with relatively shorter cell cycles favors B cell development, while the accumulation of PU.1 by a lengthened cell cycle favors macrophage development [21]. In the future, the further delineation of the molecular signaling pathway mechanism between cell cycle length and PU.1 accumulation will be a very meaningful discovery.

In morphological development, the development of Drosophila follicular epithelium anterior and posterior is exquisitely controlled by a bistable switch. Although the epidermal growth factor receptor (EGFR) receives the same signal, the developmental outcome (anterior versus posterior) might be different, depending on the equilibrium state of Midline/H15 and Mirror, which are transcription factors downstream of EGFR and form a toggle switch. In the anterior, bone morphogenetic protein (BMP) inhibits Midline/H15 and activates Mirror, whereas in the posterior, JAK/STAT inhibits Mirror and activates Midline/H15. The mutual inhibition between Midline/H15 and Mirror stabilizes their decision on tissue morphologic formation decisions [22]. In the Wnt/β-catenin signaling pathway, Wnt acts as a morphogen in tissue patterning, and glycogen synthase kinase 3 (GSK3) and the scaffold protein Axin form a positive-feedback bistable switch to control the β-catenin destruction complex in a stable off or on state [23]. A recent study searched comprehensively in the existing database SIGnaling Network Open Resource for two-node bistable structures, which constitute the smallest networks. This study revealed in theory seven common topologies of two-node bistability and their distribution characteristics in some cancers. These bistable topologies are different in robustness and stochasticity; therefore, they exert different influences upon the distribution characteristics [24].

2.3. In Multicellular Systems

Bistable switches exist at different levels of the nervous system. Purkinje neurons are important neurons in the brain that connect the cerebellar cortex to deeper structures in the cerebellum. The output of Purkinje neurons is characterized by bistable switching, namely, an abrupt transition between tonic firing and quiescence. It is partly known that this bistability involves certain ion currents and depolarizing afterpotential, which was discussed in a review article [25]. Rat experiments suggested that bistability is a necessary condition for inverse stochastic resonance (ISR), a phenomenon in which Purkinje cells are efficiently inhibited by noise of certain variances [26]. In models of the visual cortex composed of different types of GABAergic cells, bistable circuit switching can be observed from the interactions between different cell types characterized by inhibitory and disinhibitory states [27]. In the treatment of the psychiatric disorder major depressive disorder (MDD), studies have found that the hypothalamus–pituitary–adrenal (HPA) axis and central nervous system (CNS) regions such as the hippocampus inhibit each other, forming a bistable switch toggling between euthymic and depressed states. The revelation of this mechanism provides a good theoretical basis for disease treatment strategies [28].

Bistable switching has also been found in the insulin response (Figure 1d) [29,30,31]. This switching strategy is important to ensure that the body can quickly lower blood sugar without causing excessive hypoglycemia [3,32].

2.4. Variation and Extension

In addition to the classical structures and events that have been systematically studied above, the variation and extension of naturally occurring bistability make them more unique and functional.

The bistable switching of Src-family kinases (SFKs) requires no external feedback loops, owing to their closed autoinhibited conformation characterized by the interaction of the Src homology 2 domain with an inhibitory phosphotyrosine in the C-terminus [33]. In the study of GTPase, it was found that interlinked GTPase cascades consisting of two species coupled through positive and negative feedback loops create more robust bistability than that of a single species [34]. In an in vitro subclone of human marrow stromal cells, two sequentially associated bistable switches were observed. First, inhibitor pretreatment suppresses the myogenic ability of MAPK and converts them into osteogenic precursors that become responsible for osteogenic inducers. This inhibitor pretreatment has memory. Then, the MAPK inhibitor-pretreated precursors are stimulated by osteogenic inducers, exhibiting ultrasensitivity, ‘‘all-or-none’’ behavior, and memory [35]. Therefore, the sequential combination of multiple bistable switches realizes binary decisions in response to external signals during cell differentiation. However, this study only presents phenomena, and the cellular signaling or genetic regulating mechanisms behind them have not yet been revealed.

In budding yeast experiments, the bistable switch was combined with spatial regulation to enable cells to transmit intergenerational memory. The switch element Far1 translocating from nucleus to cytoplasm transmits the memory of pheromone exposure to the next generation [36]. Aurora B kinase, which is involved in cell division regulation, is located at the centromere. Its own activity forms a bistable switch with another inhibitory phosphatase, making Aurora B kinase have two different activities, high and low. What is very special is that Aurora B kinase and its substrate are separated by a long distance, especially the extension caused by cell division. This seemingly unreasonable long distance combined with the bistable switching mechanism allows Aurora B kinase to be clearly divided into two active states in the middle, accurately ensuring that after the division extends to a certain extent, the substrate’s response to Aurora B kinase is timely downregulated to complete chromosome separation [37]. In Chlamydomonas reinhardtii, a light-responsive bistable switch regulates the transition between the G1 and S/M cycles. During the day when the light is sufficient, the S/M entry threshold increases significantly, but the G1 entry threshold does not increase significantly, so the hysteresis of the switch is greatly increased, which increases the cell’s S/M cycle and the cell’s size in the G1 phase. This bistable switch acts as a light-responsive sizer [38]. Exocytosis involves forming a lipid bilayer membrane neck between the vesicle and plasma membrane. The extended membrane tubule of the neck has two stable shapes: catenoidal microtubule and cylindrical nanotubule. It is demonstrated that the bistable switch of these two states exists in macrophages and controls vesicle content release in a “kiss-and-run” mode [39].

It is important to note that living systems are dynamic, and the dynamics of the system can interact with bistable switches. For example, morphogen-controlled bistable switches in developing tissues can be significantly altered by intrinsic noise [40].

3. Applications of Bistable Switches in Synthetic Biology

3.1. Design and Basic Applications

There are two basic ways to achieve bistable switches in synthetic biology. One is at the level of genetic regulation such as mutual inhibition of gene expression, which is the most commonly used design; the other is at the level of protein activity regulation, which achieves protein activation or inactivation through modification or sealing. In addition, there are continuous research attempts to develop more bistable switch implementations. According to the large-scale simulations of two- and three-component cellular network topologies to compare the purely enzymatic networks, purely transcriptional networks, and their hybrid networks, the hybrid networks were found to be the most robust in generating bistability, while the pure transcriptional networks are the most fragile [41].

The most classic designs of bistability include an early genetic toggle switch constructed in E. coli (Figure 2a) [42]. In this design, the products of the two genes act as transcriptional inhibitors for each other. Chemical or thermal induction can flip the stable state. Compared with the controls without this toggle switch, the switch-containing system can sustain either state without continuous stimulation once induced. Many subsequent experiments and applications on bistable switches are based on this design model. In an applied study, the researchers constructed up- and down-regulated genes related to stress-induced mutagenesis in E. coli through genetic toggle switches, which acted as a stress-induced mutagenesis module for strain engineering to accelerate the adaptive evolution of microorganisms. In a stress-free environment, the cells were in a closed state of mutagenic cell response; when placed in an antibiotic stress environment, the cells switched to an open state of mutagenic cell response state and reached a very high level of antibiotic resistance [43]. Mutual inhibition of E. coli populations by opposing morphogens achieved bistable response to dynamic morphogen gradients, which is similar to the spatial information gaining during development in multicellular organisms [44]. This framework laid the foundation of both pattern creation in cell population devices and tissue differentiation engineering in organ regeneration. In addition to prokaryotes, the mutual inhibition design has also been implemented in mammalian cells. Two different antibiotics were used to control the transcription of two mutually repressive genes. This bistable switch was extremely stable in mammalian cells: locking in an antibiotic for three days allowed the cells to stably switch to a stable state for up to three weeks. The stable cell line _TOGGLECHO₃₇ cells were also implanted into the peritoneal cavity of mice to achieve the control of the synthetic bistable switch in vivo, pointing out an optimistic direction for its application in clinical diagnosis or treatment [45].

In addition to the widely used mutual repression of gene expression, there are more complex designs of bistable switches to solve some specific problems or adapt to specific usage environments. In a study, a positive feedback loop was constructed in Saccharomyces cerevisiae by means of genetically autocatalytic expression, and the all-or-none bistable switch of the reporter gene was realized through adjusting parameters by computation and experiment (Figure 2b-1). This method of implementing bistable switches is different from genetically mutual repression. On the one hand, the on-state in the experiment presents under the continuous presence of the inducer, and its hysteresis and irreversibility still need to be confirmed by data. On the other hand, it has randomness in transition and time, unlike the overall consistency of the population of mutual repression, but it can control the proportion of switching cells by adjusting parameters. Moreover, the cells in the off-state always maintain higher division and proliferation activity than those in the on-state, tending to replenish and maintain the population size before the switch. The researchers compared it to the differentiation of multicellular organisms, where a fixed proportion of high-state cells are randomly generated from a homogeneous pool of precursors to differentiate, while the pool divides and renews to maintain a stable number of precursors. This study provides important inspiration for applying synthetic bistable switches to the regulation of cell differentiation [46]. Similarly, a positive feedback loop of a transcription factor connected to a growth differentiation gene was built in Saccharomyces cerevisiae to achieve a bistable switch that controls the rate of yeast growth (Figure 2b-2) [47]. In mammalian cells, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability. To solve this problem, the researchers introduced a positive feedback loop into the HEK293T cell line, allowing the activator and repressor to compete for binding to the same DNA operator site, thereby achieving a bistable switch. This design has provided a reference for the application of monomeric DNA-binding domains such as CRISPR in mammalian cells [48]. In a study to control cellular uptake, researchers coupled enzyme activity with a gene regulatory circuit to shape the uptake response as a bistable switch in bacteria (Figure 2c) [49]. Combining homodimerization and the cooperative binding of the transcription factor to the promoter is another strategy to generate robust bistability [50]. Sequestration combined with positive feedback has also been demonstrated to achieve a bistable switch in E. coli (Figure 2d) [51,52]. Another breakthrough design is a protein-phosphorylation toggle network that is completely independent of genetic regulation. This design was implemented in Saccharomyces cerevisiae, which realized mutual cross-repression at protein activity level through protein–protein phosphorylation interactions (Figure 2e). The complex bistable switch network is built from 11 phospho-in and phospho-out signal transduction elements. Multiple nodes provided more possibilities for fine-tuning. Regulation at the protein activity level showed great advantage and fast response. In this design, the output is the translocation of fluorescent protein between the nucleus and cytoplasm, which can fully respond to the switch within minutes [53].

3.2. Expansion of Application

Bistable switches have greater application potential when combined with other regulations or methodologies (Figure 3). The naturally occurring mutual inhibiting CI/Cro genetic switch in bacteriophage λ mentioned in Section 2.1 was adopted in engineered E. coli to sense and record antibiotic exposure during passage through the mouse gut. This bistable switch functioned as a memory system in which time-limited anhydrotetracycline administration switched the bacteria to the Cro state for many divisions. Without administration, the bacteria stayed in the CI state stably [54]. This work shows the diagnostic and therapeutic potential of the switch. A synthetic bistable switch can be used as a programmable sequential logic to control a cell’s structural basis [55]. Combining a bistable switch and a repressilator within the AC–DC circuit generates novel dynamical behaviors such as control of oscillation coherence and spatial signal propagation [56]. Some bistable switch structures can generate oscillation by adjusting parameters and changing external signals [57]. Based on this, a study rewired in yeast cells the endogenous bistable switch that determines two different fates of cell aging, reconstructed a synthetic circuit that can be adjusted to oscillate, slowed down any form of aging of yeast cells, and extended the lifespan of yeast cells [58]. Some studies suggested that a synthetic bistable switch combined with diffusible molecules can form a stable pattern-forming system [59]. A study used a genetic toggle switch in E. coli and demonstrated through quantitative measurement and mathematical reconstruction that hysteresis, position, timing, and boundaries of the pattern formation can be precisely controlled, providing an experimental and theoretical basis for its application in engineered biomaterials, detection, and clinical diagnosis [60]. Red/far-red light-responsive bistable toggle switch controlling gene expression is a highly practical design that experimentally realized spatially controlled engineering of angiogenesis in chicken embryos. It is also compatible with different mammalian cell lines, including human primary cells [61,62], which have high clinical application development value. In addition to cellular systems, cell-free synthetic bistable systems have also been developed. A genetic mutual inhibition bistable switch has been successfully built via the cell-free protein synthesis (CFPS) technology [63]. In another research study, researchers have set up a bistable autoregulatory circuit by using four synthetic DNA strands and three essential enzymes in vitro [64]. The cell-free synthetic platforms broaden the future application scenarios for bistable switches.

Large-scale analysis of bistable networks is considered to be an idea for finding cancer drug targets or diagnosis biomarkers [65]. A research group has developed a top-down mining approach to explore gene switches on a genome-scale level. Through analysis of human breast cancer microarray data, they identified bimodality within the cancer samples and between cancer and non-cancer samples and found potential biomarker and drug therapy targets for breast cancer [66]. Because bistable switching is an important regulatory mechanism of cellular differentiation, including transcription factor cross-repression, another research group has tried to use this as a basis and designed a computational methodology for screening and predicting cellular reprogramming in medicine and basic research [67].

3.3. Improving the Performance of Bistable Switches

Computationally, more comprehensive and in-depth models help build a stable and accurate bistable switch, greatly reducing blind trials and failures in experiments. Quantifying the translation strength of the ribosome binding site (RBS) is an approach to help design genetic toggle switches with predetermined bistability, based on which a biophysical method has been developed [68]. Adjacent transcriptional regions (ATRs), including GC content, size, and stability of mRNA folding near ribosomal binding sites, have different effects on gene expression. Thus, quantification of these effects could help to design and tune the gene expression to construct a bistable switch [69]. Circuit–host interactions are important factors affecting the success and precision of synthetic switching. The bistable switch could be context-dependent if the system has scarce resource competitions or promoter leakiness [70,71]. In computational models, it is predicted that growth feedback significantly changes the qualitative states [72]. However, the bistable switch is also refractory to growth-mediated dilution and retrieves its memory after the fast-growth phase [73]. Therefore, in experimental design, the dynamic effect of cell growth on switching should be considered, and reliable data should be collected during the period when the two are in equilibrium. Because loads bias genetic and signaling switches in synthetic systems, it is necessary to incorporate the effects of downstream components [74]. For the bistable switch based on reversible covalent modification (RCM), the researchers revealed its potential in generating ultrasensitive responses and provided a clear theoretical basis for its application [75].

At the operational level, some current research is gradually addressing experimental technical barriers and realizing more possibilities. CRISPRi has been a powerful tool for creating synthetic gene circuits, but its potential is limited in constructing dynamical and multistable synthetic circuits due to the lack of cooperation. This problem is now solved by constructing two nodes producing sgRNAs that repress each other. A CRISPRi bistable switch has been demonstrated in E. coli populations. This method also supports the construction of CRISPRi multistable switches [76]. Based on the successful construction of bistable switches, some researchers are exploring to synthesize multistable control beyond bistability in mammalian cells by combining positive autoregulation with cross-inhibition [77]. Regarding precision, a study has shown that gene copy number variation brings non-negligible qualitative shifts to the bistable switch [78]. Through converting a multi-copy genetic toggle switch into a single-copy circuit in E. coli, the researchers increased the bistable robustness and placed minimal metabolic burden on the host cell [79].

Bistable switching has also been shown to be reflected in the communication and balance of cellular populations, including the maintenance of phenotypic consistency in single-strain microbial communities [80], interacting between strains [81], controlling cell-type ratios in microbial consortia [82], and tuning phenotypic diversification in multicellular organisms [83]. For cell populations, the states could be reset and maintained in an undecided state by periodic perturbations [84]. The study of bistable switches in cell populations has provided an important experimental and computational basis for the design of synthetic microbial consortia and tissue engineering.

4. Perspectives

This article reviews bistable switches from two aspects: natural life and synthetic biology applications. The comparison between the two (

Table 1. Comparisons between naturally occurring and synthetic bistable switches.

	Natural Bistable Switches	Synthetic Bistable Switches	Main Differences
Structural functional basis	1. Protein–protein interaction. 2. Genetic regulation.	1. Genetic regulation (primary). 2. Protein–protein regulation.	In natural life, bistable switches based on protein–protein interactions are very common; in synthetic biology, most bistable switches are currently implemented through genetic regulation.
Topology complexities	1. Ranging from single node (autocatalytic) to network, with network being the most common. 2. Usually composed of multiple positive and negative pieces of feedback. 3. Superimposed by multiple switches.	1. Most of them use the mode of two-node mutual repression. 2. Mostly use positive feedback, but rarely add negative feedback. 3. The superimposition of multiple switches is not yet implemented.	The complexity of natural bistable switches is much higher than that of synthetic bistable switches.
Adjustment levels	1. Acellular structure (e.g., viruses). 2. Intracellular signaling pathway. 3. Multicellular colonies (e.g., bacterial colonies). 4. Organismal development in multicellular organisms. 5. Individual systems of multicellular organisms.	1. Individual systems of multicellular organisms. 2. Multicellular populations. 3. Organismal development in multicellular organisms.	The regulation scope of natural bistable switches is much wider than that of current synthetic bistable switches.
Functions	1. Making a unified response to external signals. 2. Maintaining the stability of life activities. 3. Memory or irreversibility.	1. Memory or irreversibility 2. Regulation of the function or state of cells, individual organisms, or groups. 3. Building new functions into organisms.	The application of synthetic bistable switches focuses on further regulating and changing the functional state of organisms, or giving organisms additional new functions.

) clearly shows that bistable switches are skillfully and widely used in natural life, while they are still in the early stages of exploration and experimentation in synthetic biology applications. Their design principles, implementation methods, means of regulation, and application scenarios undergo a process that requires creative development. At the same time, this comparison also shows that bistable switches in natural life are textbooks and material libraries for synthetic biology applications. Researchers’ discovery, imitation, and transformation of bistable switches in natural life have promoted the development of synthetic biology applications.

In natural life systems, from the simplest non-cellular structures such as viruses to prokaryotic cells, to signaling pathways within eukaryotic cells, and to the systems of multicellular organisms, all are regulated by bistable switches. These bistable switches generally contain a large number of components with complex relationships between them. Even for viruses that do not have a cellular structure and prokaryotes with a simple cell structure, the revelation of bistable switches in their life activities is still just the tip of the iceberg. For example, in the lysis–lysogeny decision of phage λ and lac operon mentioned above, although the bistable switch and some key components were discovered decades ago, computational models are still being constructed and optimized to this day, and the regulatory elements involved and their functions are still being discovered in experiments [6,85]. The development and regulation of eukaryotic organisms, from the inside of cells to between cells and even the entire organism, are even more complex. For example, the cell cycle and MAPK signaling, which are the most classic and systematically studied, have benefited from the continuous in-depth revelation of their molecular mechanisms, and the computational model of the bistable switch has been continuously updated and improved. They each act as an independent bistable switch, involving a combination of multiple positive and negative pieces of feedback in the cellular event regulation network [13]. Furthermore, positive feedback is formed between the two bistable switches [20]. At a higher level, the combined regulation of multiple bistable switches has also been observed in experiments, such as the effects of the sequential regulation of MAPK inhibition and osteogenic induction on the differentiation of marrow stromal cells mentioned above [35]. For these complex bistable switches, the molecular mechanisms remain to be revealed. From the analysis of the cellular signaling network, one sees that the simplest two-node bistability has at least seven common topologies [24], which shows the broadness of the possibilities and richness of multi-node bistable switches in the complex cellular network. In the macroscopic systems of multicellular organisms, such as the nervous system and hormone response, we have observed macroscopic bistable switch phenomena [25,27,28,31]. On the one hand, these phenomena can be simulated by computational models, but on the other hand, we should realize that such stable macroscopic phenomena are fundamentally regulated by larger microscopic systems. This level is something that artificial synthetic biology has not yet reached.

The artificial biosynthesis of bistable switches began with the creation of a pair of successful transcriptionally mutual repressors in E. coli more than 20 years ago [42]. Many subsequent designs also follow this design principle, which has two advantages. First, with only two nodes, the structure is simple, making experimental construction and parameter adjustment convenient. Second, the regulation of gene transcription is more direct and stable than other methods, such as enzyme activity regulation. However, this is also its disadvantage. The simple structure means that it is difficult to expand to form a complex or advanced regulatory network; transcriptional regulation requires a long response time, which is difficult for timely response and real-time functioning. More construction modes for bistable switches could fill these gaps. One of the above-mentioned studies designed a circuit with up to 11 nodes and transmitted signals by modifying proteins in the nodes while solving the two drawbacks of the design of transcriptionally mutual repressors [53]. This is just one of the countless possible designs. Just as there are thousands of bistable switch configuration patterns in nature, richer, more efficient, and more versatile designs will be a research trend in the future. In addition to independently constructing a bistable switch circuit into the cell to realize new functions, coupling the bistable switch to a signaling pathway of the cell itself to regulate existing cell functions is also a research direction with particularly promising application prospects. For example, there are some preliminary application studies on regulating cell differentiation [62] and aging [58]. At present, this type of research still has a long way to go before clinical application, and most of it is still in the experimental stage of prokaryotic cells, yeasts, or other in vitro systems. On the one hand, the experimental construction of the bistable switch requires the support of continuously optimized innovative molecular biology technologies to break through operational technical barriers, such as the use and modification of CRISPR technology; on the other hand, computational models are needed to provide parameter adjustment ranges, evaluate or increase the system’s anti-interference stability, and even assist in the design of the bistable switch. Therefore, the optimization of molecular biology experimental techniques and computational models will greatly promote the application of bistable switches. In addition, based on the existing databases of genomes, transcriptomes, proteomes, etc., establishing algorithms to analyze the bistable switch characteristics of these data will be of great practical value for clinical diagnosis of diseases, finding therapeutic targets, and judging prognosis. This is a research direction that is currently closer to the clinic.

From bistable switches in natural life systems to those in artificial biosynthesis applications, we have seen huge differences in their complexity and sophistication, as well as great prospects for artificial biosynthesis applications. Artificial biosynthesis applications can continue to make greater breakthroughs in designing circuit patterns that meet more needs, creating new biological functions, regulating existing functions, developing new molecular biology experimental techniques, optimizing and innovating computational models, and analyzing bistable switches in large-scale data. At the same time, the continued exploration of bistable switches in natural life systems also provides a deeper theoretical foundation for artificial biosynthesis applications, further promoting their development.