Synthetic Gene Circuits Research Articles

Nonmonotone invasion landscape by noise-aware control of metastasis activator levels

A major pharmacological assumption is that lowering disease-promoting protein levels is generally beneficial. For example, inhibiting metastasis activator BACH1 is proposed to decrease cancer metastases. Testing such assumptions requires approaches to measure disease phenotypes while precisely adjusting disease-promoting protein levels. Here we developed a two-step strategy to integrate protein-level tuning, noise-aware synthetic gene circuits into a well-defined human genomic safe harbor locus. Unexpectedly, engineered MDA-MB-231 metastatic human breast cancer cells become more, then less and then more invasive as we tune BACH1 levels up, irrespective of the native BACH1. BACH1 expression shifts in invading cells, and expression of BACH1ʼs transcriptional targets confirm BACH1ʼs nonmonotone phenotypic and regulatory effects. Thus, chemical inhibition of BACH1 could have unwanted effects on invasion. Additionally, BACH1ʼs expression variability aids invasion at high BACH1 expression. Overall, precisely engineered, noise-aware protein-level control is necessary and important to unravel disease effects of genes to improve clinical drug efficacy.

The design of synthetic gene circuits in plants: new components, old challenges.

The fascination produced by the possibility of engineering plants with augmented capabilities has accompanied plant biotechnology since its origins. This prospect has become even more relevant in present times under the pressure imposed by climate change and population growth. Today´s plant biotechnologists appraise this challenge with the tools of synthetic biology, which facilitate the assembly of synthetic gene circuits (SGCs) from their modular components. Transcriptional SGCs take environmental or endogenous inputs and operate them using transcriptional signals in ways that do not necessarily occur in nature, generating new physiological outputs. Many genetic components have been developed over the years that can be employed in the design and construction of plant SGCs. This review aims to provide an updated and categorized view of the components available, proposing a general scheme that facilitates the classification of circuit components in sensor, processor, and actuator modules. Following this analogy, we review the latest advances in the design of SGC and discuss the main challenges ahead.

Automated design of gene circuits with optimal mushroom-bifurcation behavior

Recent advances in synthetic biology are enabling exciting technologies, including the next generation of biosensors, the rational design of cell memory, modulated synthetic cell differentiation, and generic multifunctional biocircuits. These novel applications require the design of gene circuits leading to sophisticated behaviors and functionalities. At the same time, designs need to be kept minimal to avoid compromising cell viability. Bifurcation theory addresses such challenges by associating circuit dynamical properties with molecular details of its design. Nevertheless, incorporating bifurcation analysis into automated design processes has not been accomplished yet. This work presents an optimization-based method for the automated design of synthetic gene circuits with specified bifurcation diagrams that employ minimal network topologies. Using this approach, we designed circuits exhibiting the mushroom bifurcation, distilled the most robust topologies, and explored its multifunctional behavior. We then outline potential applications in biosensors, memory devices, and synthetic cell differentiation.

A Clinically Viable Approach to Cell Programming

A Clinically Viable Approach to Cell Programming

Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression.

Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5'-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5' end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.

CRISPR-associated type V proteins as a tool for controlling mRNA stability in S. cerevisiae synthetic gene circuits.

Type V-A CRISPR-(d)Cas system has been used in multiplex genome editing and transcription regulation in both eukaryotes and prokaryotes. However, mRNA degradation through the endonuclease activity of Cas12a has never been studied. In this work, we present an efficient and powerful tool to induce mRNA degradation in the yeast Saccharomyces cerevisiae via the catalytic activity of (d)Cas12a on pre-crRNA structure. Our results point out that dFnCas12a, (d)LbCas12a, denAsCas12a and two variants (which carry either NLSs or NESs) perform significant mRNA degradation upon insertion of pre-crRNA fragments into the 5'- or 3' UTR of the target mRNA. The tool worked well with two more Cas12 proteins-(d)MbCas12a and Casϕ2-whereas failed by using type VI LwaCas13a, which further highlights the great potential of type V-A Cas proteins in yeast. We applied our tool to the construction of Boolean NOT, NAND, and IMPLY gates, whose logic operations are fully based on the control of the degradation of the mRNA encoding for a reporter protein. Compared to other methods for the regulation of mRNA stability in yeast synthetic gene circuits (such as RNAi and riboswitches/ribozymes), our system is far easier to engineer and ensure very high performance.

A data-driven optimization method for coarse-graining gene regulatory networks

One major challenge in systems biology is to understand how various genes in a gene regulatory network (GRN) collectively perform their functions and control network dynamics. This task becomes extremely hard to tackle in the case of large networks with hundreds of genes and edges, many of which have redundant regulatory roles and functions. The existing methods for model reduction usually require the detailed mathematical description of dynamical systems and their corresponding kinetic parameters, which are often not available. Here, we present a data-driven method for coarse-graining large GRNs, named SacoGraci, using ensemble-based mathematical modeling, dimensionality reduction, and gene circuit optimization by Markov Chain Monte Carlo methods. SacoGraci requires network topology as the only input and is robust against errors in GRNs. We benchmark and demonstrate its usage with synthetic, literature-based, and bioinformatics-derived GRNs. We hope SacoGraci will enhance our ability to model the gene regulation of complex biological systems.

Rational design of functional amyloid fibrillar assemblies.

Amyloid fibrillar assemblies, originally identified as pathological entities in neurodegenerative diseases, have been widely adopted by various proteins to fulfill diverse biological functions in living organisms. Due to their unique features, such as hierarchical assembly, exceptional mechanical properties, environmental stability, and self-healing properties, amyloid fibrillar assemblies have been employed as functional materials in numerous applications. Recently, with the rapid advancement in synthetic biology and structural biology tools, new trends in the functional design of amyloid fibrillar assemblies have begun to emerge. In this review, we provide a comprehensive overview of the design principles for functional amyloid fibrillar assemblies from an engineering perspective, as well as through the lens of structural insights. Initially, we introduce the fundamental structural configurations of amyloid assemblies and highlight the functions of representative examples. We then focus on the underlying design principles of two prevalent strategies for the design of functional amyloid fibrillar assemblies: (1) introducing new functions via protein modular design and/or hybridization, with typical applications encompassing catalysis, virus disinfection, biomimetic mineralization, bio-imaging, and biotherapy; and (2) dynamically regulating living amyloid fibrillar assemblies using synthetic gene circuits, with typical applications in pattern formation, leakage repair, and pressure sensing. Next, we summarize how breakthroughs in characterization techniques have contributed to unveiling the structural polymorphism of amyloid fibrils at the atomic level, and further clarifying the highly diverse regulation mechanisms of amyloid fibrillar assembly and disassembly fine-tuned by various factors. The structural knowledge may significantly aid in the structure-guided design of amyloid fibrillar assemblies with diverse bio-activities and adjustable regulatory properties. Finally, we envision that a new trend in functional amyloid design may emerge by integrating structural tunability, synthetic biology and artificial intelligence.

Multidimensional control of therapeutic human cell function with synthetic gene circuits

Synthetic gene circuits that precisely control human cell function could expand the capabilities of gene- and cell-based therapies. However, platforms for developing circuits in primary human cells that drive robust functional changes in vivo and have compositions suitable for clinical use are lacking. Here, we developed synthetic zinc finger transcription regulators (synZiFTRs), which are compact and based largely on human-derived proteins. As a proof of principle, we engineered gene switches and circuits that allow precise, user-defined control over therapeutically relevant genes in primary T cells using orthogonal, US Food and Drug Administration-approved small-molecule inducers. Our circuits can instruct T cells to sequentially activate multiple cellular programs such as proliferation and antitumor activity to drive synergistic therapeutic responses. This platform should accelerate the development and clinical translation of synthetic gene circuits in diverse human cell types and contexts.

Bacillus integrative plasmid system combining a synthetic gene circuit for efficient genetic modifications of undomesticated Bacillus strains

BackgroundOwing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems.ResultsHere, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains.ConclusionThe Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

Negatively Competitive Incoherent Feedforward Loops Mitigate Winner-Take-All Resource Competition.

The effects of host resource limitations on the function of synthetic gene circuits have gained significant attention over the past years. Hosts, having evolved resource capacities optimal for their own genome, have been repeatedly demonstrated to suffer from the added burden of synthetic genetic programs, which may in return pose deleterious effects on the circuit's function. Three resource controller archetypes have been proposed previously to mitigate resource distribution problems in dynamic circuits: the local controller, the global controller, and a "negatively competitive" regulatory (NCR) controller that utilizes synthetic competition to combat resource competition. The dynamics of negative feedback forms of these controllers have been previously investigated, and here we extend the analysis of these resource allocation strategies to the incoherent feedforward loop (iFFL) topology. We demonstrate that the three iFFL controllers can attenuate Winner-Take-All resource competition between two bistable switches. We uncover that the parameters associated with the synthetic competition in the NCR iFFL controller are paramount to its increased efficacy over the local controller type, while the global controllers demonstrate to be relatively ineffectual. Interestingly, unlike the negative feedback counterpart topologies, iFFL controllers exhibit a unique coupling of switch activation thresholds which we term the "coactivation threshold shift" effect. Finally, we demonstrate that a nearly fully orthogonal set of bistable switches could be achieved by pairing an NCR controller with an appropriate level of controller resource consumption.

Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type‐ and dose‐specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND‐like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine‐tuned levels of output proteins using an miRNA‐mediated output fine‐tuning technology (miSFITs). Specifically, we created an “hPSC ON” circuit using a model‐guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC‐specific detection and graded output protein production. Next, we used an empirical approach to create an “hPSC‐Off” circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state‐specific and fine‐tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state‐specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC‐derived tissues and beyond.

Agent-Based Modeling of Microbial Communities.

Microbial communities are complex living systems that populate the planet with diverse functions and are increasingly harnessed for practical human needs. To deepen the fundamental understanding of their organization and functioning as well as to facilitate their engineering for applications, mathematical modeling has played an increasingly important role. Agent-based models represent a class of powerful quantitative frameworks for investigating microbial communities because of their individualistic nature in describing cells, mechanistic characterization of molecular and cellular processes, and intrinsic ability to produce emergent system properties. This review presents a comprehensive overview of recent advances in agent-based modeling of microbial communities. It surveys the state-of-the-art algorithms employed to simulate intracellular biomolecular events, single-cell behaviors, intercellular interactions, and interactions between cells and their environments that collectively serve as the driving forces of community behaviors. It also highlights three lines of applications of agent-based modeling, namely, the elucidation of microbial range expansion and colony ecology, the design of synthetic gene circuits and microbial populations for desired behaviors, and the characterization of biofilm formation and dispersal. The review concludes with a discussion of existing challenges, including the computational cost of the modeling, and potential mitigation strategies.

Two-Layered Microfluidic Devices for High-Throughput Dynamic Analysis of Synthetic Gene Circuits in E. coli.

Escherichia coli is a common chassis for synthetic gene circuit studies. In addition to the dose-response of synthetic gene circuits, the analysis of dynamic responses is also an important part of the future design of more complicated synthetic systems. Recently, microfluidic-based methods have been widely used for the analysis of gene expression dynamics. Here, we established a two-layered microfluidic platform for the systematic characterization of synthetic gene circuits (eight strains in eight different culture environments could be observed simultaneously with a 5 min time resolution). With this platform, both dose responses and dynamic responses with a high temporal resolution could be easily derived for further analysis. A controlled environment ensures the stability of the bacterial growth rate, excluding changes in gene expression dynamics caused by changes of the growth dilution rate. The precise environmental switch and automatic micrograph shooting ensured that there was nearly no time lag between the inducer addition and the data recording. We studied four four-node incoherent-feedforward-loop (IFFL) networks with different operators using this device. The experimental results showed that as the effect of inhibition increased, two of the IFFL networks generated pulselike dynamic gene expressions in the range of the inducer concentrations, which was different from the dynamics of the two other circuits with only a simple pattern of rising to the platform. Through fitting the dose-response curves and the dynamic response curves, corresponding parameters were derived and introduced to a simple model that could qualitatively explain the generation of pulse dynamics.

E. coli biosensor based on modular GFP and luxI/luxR cyclic amplification circuit for sensitive detection of lysine.

In this study, an E. coli biosensor based on modular green fluorescent protein and luxI/IuxR cycle amplification circuit was constructed for sensitive detection of bioavailable lysine. The results indicated that the luxI/IuxR positive feedback circuit based on quorum sensing can be used as a signal amplifier to improve the sensitivity to lysine detection with the detection limit of 256 nM. The presented method was more sensitive than the previously reported whole-cell fluorescent microbial biosensors. In addition, the developed E. coli biosensor was specific for lysine detection, and other amino acids and proteins did not cause any interference. The constructed genetic engineered biosensor was accurate for lysine detection, the lysine content of 6.87 ± 0.36% in tryptone was successfully measured, and after adding 10, 30, and 50 μM lysine in tryptone, the recoveries of 109.98 ± 10.44%, 103.88 ± 7.66%, and 105.89 ± 6.34% were obtained, respectively. Furthermore, as the design of the genetic engineered biosensor is modular, it can conceivably be utilized as a component in the design of more complex synthetic gene circuits without any changes to the amplifier and reporter system.

A sensitive and specific nano-vehicle based on self-amplified dual-input synthetic gene circuit for intracellular imaging and treatment

Synthetic genetic circuits (SGCs) that sense multiple biomarkers and respond intelligently provide a powerful tool for intracellular biosensing. The SGC is usually loaded into the nanoscale liposomes to build functional intracellular nano-vehicles, widely applied in diagnosing and treating diseases. However, because the system needs to identify multiple targets to activate, the sensitivity will be inevitably reduced though the specificity is improved, leading to false-negative results in diagnosis and low killing dosage in treatment. Such compromise between specificity and sensitivity has been a bottleneck problem for the field. We innovatively invented the self-amplified dual-input (SADI) SGC@liposome nano-vehicle and broke the bottleneck problem above. It provides multiple sites for regulating sensitivity at both coarse and fine levels, allowing researchers to conveniently balance the sensitivity and specificity according to the application and instrumental setups. In recognizing ovarian cancer cells, the nano-vehicle could enhance the sensitivity by nearly 10-fold, and the specificity remained at high levels of 16-fold. We also changed the output fluorescent signal to output effectors such as apoptosis regulator (BAX) and proliferation-inhibiting protein (p21) and demonstrated the application range. Furthermore, we verified the generality of the system by applying it to target different cells. We believe it will provide a convenient and powerful tool for biosensors and targeted therapy.

Synthetic neuromorphic computing in living cells

Computational properties of neuronal networks have been applied to computing systems using simplified models comprising repeated connected nodes, e.g., perceptrons, with decision-making capabilities and flexible weighted links. Analogously to their revolutionary impact on computing, neuro-inspired models can transform synthetic gene circuit design in a manner that is reliable, efficient in resource utilization, and readily reconfigurable for different tasks. To this end, we introduce the perceptgene, a perceptron that computes in the logarithmic domain, which enables efficient implementation of artificial neural networks in Escherichia coli cells. We successfully modify perceptgene parameters to create devices that encode a minimum, maximum, and average of analog inputs. With these devices, we create multi-layer perceptgene circuits that compute a soft majority function, perform an analog-to-digital conversion, and implement a ternary switch. We also create a programmable perceptgene circuit whose computation can be modified from OR to AND logic using small molecule induction. Finally, we show that our approach enables circuit optimization via artificial intelligence algorithms.

Emergence of qualitative states in synthetic circuits driven by ultrasensitive growth feedback.

The mutual interactions between the synthetic gene circuits and the host growth could cause unexpected outcomes in the dynamical behaviors of the circuits. However, how the steady states and the stabilities of the gene circuits are affected by host cell growth is not fully understood. Here, we developed a mathematical model for nonlinear growth feedback based on published experimental data. The model analysis predicts that growth feedback could significantly change the qualitative states of the system. Bistability could emerge in a circuit without positive feedback, and high-order multistability (three or more steady states) arises in the self-activation and toggle switch circuits. Our results provide insight into the potential effects of ultrasensitive growth feedback on the emergence of qualitative states in synthetic circuits and the corresponding underlying mechanism.

Steady-State Operation of a Cell-Free Genetic Band-Detection Circuit.

Pattern formation processes play a decisive role during embryogenesis and involve the precise spatial and temporal orchestration of intricate gene regulatory processes. Synthetic gene circuits modeled after their biological counterparts can be used to investigate such processes under well-controlled conditions and may, in the future, be utilized for autonomous position determination in synthetic biological materials. Here, we investigated a three-node feed-forward gene regulatory circuit in vitro that generates three distinct fluorescent outputs in response to varying concentrations of a single externally supplied morphogen. The circuit acts as a band detector for the morphogen concentration and, in a spatial context, could serve as a stripe-forming gene circuit. We simulated the behavior of the genetic circuit in the presence of a morphogen gradient using a system of ordinary differential equations and determined optimal parameters for stripe-pattern formation using an evolutionary algorithm. To analyze the subcircuits of the system, we conducted cell-free characterization experiments and finally tested the whole genetic circuit in nanoliter-scale microfluidic flow reactors that provided a continuous supply of cell extract and metabolites and allowed removal of degradation products. To make use of the widely employed promoters PLlacO-1 and PLtetO-1 in our design, we removed LacI from our bacterial cell extract by genome engineering Escherichia coli using a pORTMAGE workflow. Our results show that the band-detector works as designed when operated out of equilibrium within the flow reactors. On the other hand, subcircuits of the system and the whole circuit fail to generate the desired gene expression response when operated in a closed reactor. Our work thus underlines the importance of out-of-equilibrium operation of complex gene circuits, which cannot settle to a steady-state expression pattern within the finite lifetime of a cell-free expression system.