Synthetic gene circuit evolution: Insights and opportunities at the mid-scale.

Directed evolution focuses on optimizing single genetic components for predefined engineering goals by artificial mutagenesis and selection. In contrast, experimental evolution studies the adaptation of entire genomes in serially propagated cell populations, to provide an experimental basis for evolutionary theory. There is a relatively unexplored gap at the middle ground between these two techniques, to evolve in vivo entire synthetic gene circuits with nontrivial dynamic function instead of single parts or whole genomes. We discuss the requirements for such mid-scale evolution, with hypothetical examples for evolving synthetic gene circuits by appropriate selection and targeted shuffling of a seed set of genetic components in vivo. Implementing similar methods should aid the rapid generation, functionalization, and optimization of synthetic gene circuits in various organisms and environments, accelerating both the development of biomedical and technological applications and the understanding of principles guiding regulatory network evolution.

Sonogenetics-controlled synthetic designer cells for cancer therapy in tumor mouse models.

Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate the local expression and release of therapeutic cargoes. Rapid advances in remote-control technologies have enabled precise control of biological processes in time and space. We developed therapeutically active engineered bacteria mediated by a sono-activatable integrated gene circuit based on the thermosensitive transcriptional repressor TlpA39. Through promoter engineering and ribosome binding site screening, we achieved ultrasound (US)-induced protein expression and secretion in engineered bacteria with minimal noise and high induction efficiency. Specifically, delivered either intratumorally or intravenously, engineered bacteria colonizing tumors suppressed tumor growth through US-irradiation-induced release of the apoptotic protein azurin and an immune checkpoint inhibitor, a nanobody targeting programmed death-ligand 1, in different tumor mouse models. Beyond developing safe and high-performance designer bacteria for tumor therapy, our study illustrates a sonogenetics-controlled therapeutic platform that can be harnessed for bacteria-based precision medicine.

In Vitro Generation of Megakaryocytes from Engineered Mouse Embryonic Stem Cells.

The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase-mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.

A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Engineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics.

Synthetic Gene Circuits Combining CRISPR Interference and CRISPR Activation in E. coli: Importance of Equal Guide RNA Binding Affinities to Avoid Context-Dependent Effects.

Gene expression control based on clustered regularly interspaced short palindromic repeats (CRISPR) has emerged as a powerful approach for constructing synthetic gene circuits. While the use of CRISPR interference (CRISPRi) is already well-established in prokaryotic circuits, CRISPR activation (CRISPRa) is less mature, and a combination of the two in the same circuits is only just emerging. Here, we report that combining CRISPRi with SoxS-based CRISPRa in Escherichia coli can lead to context-dependent effects due to different affinities in the formation of CRISPRa and CRISPRi complexes, resulting in loss of predictable behavior. We show that this effect can be avoided by using the same scaffold guide RNA structure for both complexes.

Spatial biology of Ising-like synthetic genetic networks.

BACKGROUND: Understanding how spatial patterns of gene expression emerge from the interaction of individual gene networks is a fundamental challenge in biology. Developing a synthetic experimental system with a common theoretical framework that captures the emergence of short- and long-range spatial correlations (and anti-correlations) from interacting gene networks could serve to uncover generic scaling properties of these ubiquitous phenomena. RESULTS: Here, we combine synthetic biology, statistical mechanics models, and computational simulations to study the spatial behavior of synthetic gene networks (SGNs) in Escherichia coli quasi-2D colonies growing on hard agar surfaces. Guided by the combined mechanisms of the contact process lattice simulation and two-dimensional Ising model (CPIM), we describe the spatial behavior of bi-stable and chemically coupled SGNs that self-organize into patterns of long-range correlations with power-law scaling or short-range anti-correlations. These patterns, resembling ferromagnetic and anti-ferromagnetic configurations of the Ising model near critical points, maintain their scaling properties upon changes in growth rate and cell shape. CONCLUSIONS: Our findings shed light on the spatial biology of coupled and bistable gene networks in growing cell populations. This emergent spatial behavior could provide insights into the study and engineering of self-organizing gene patterns in eukaryotic tissues and bacterial consortia.

Algorithmic Programming of Sequential Logic and Genetic Circuits for Recording Biochemical Concentration in a Probiotic Bacterium.

Through the implementation of designable genetic circuits, engineered probiotic microorganisms could be used as noninvasive diagnostic tools for the gastrointestinal tract. For these living cells to report detected biomarkers or signals after exiting the gut, the genetic circuits must be able to record these signals by using genetically encoded memory. Complex memory register circuits could enable multiplex interrogation of biomarkers and signals. A theory-based approach to create genetic circuits containing memory, known as sequential logic circuits, was previously established for a model laboratory strain of Escherichia coli, yet how circuit component performance varies for nonmodel and clinically relevant bacterial strains is poorly understood. Here, we develop a scalable computational approach to design robust sequential logic circuits in probiotic strain Escherichia coli Nissle 1917 (EcN). In this work, we used TetR-family transcriptional repressors to build genetic logic gates that can be composed into sequential logic circuits, along with a set of engineered sensors relevant for use in the gut environment. Using standard methods, 16 genetic NOT gates and nine sensors were experimentally characterized in EcN. These data were used to design and predict the performance of circuit designs. We present a set of genetic circuits encoding both combinational logic and sequential logic and show that the circuit outputs are in close agreement with our quantitative predictions from the design algorithm. Furthermore, we demonstrate an analog-like concentration recording circuit that detects and reports three input concentration ranges of a biochemical signal using sequential logic.

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 approach 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 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 SGCs and discuss the main challenges ahead.

Performance Prediction of Fundamental Transcriptional Programs.

Transcriptional programming leverages systems of engineered transcription factors to impart decision-making (e.g., Boolean logic) in chassis cells. The number of components used to construct said decision-making systems is rapidly increasing, making an exhaustive experimental evaluation of iterations of biological circuits impractical. Accordingly, we posited that a predictive tool is needed to guide and accelerate the design of transcriptional programs. The work described here involves the development and experimental characterization of a large collection of network-capable single-INPUT logical operations─i.e., engineered BUFFER (repressor) and engineered NOT (antirepressor) logical operations. Using this single-INPUT data and developed metrology, we were able to model and predict the performances of all fundamental two-INPUT compressed logical operations (i.e., compressed AND gates and compressed NOR gates). In addition, we were able to model and predict the performance of compressed mixed phenotype logical operations (A NIMPLY B gates and complementary B NIMPLY A gates). These results demonstrate that single-INPUT data is sufficient to accurately predict both the qualitative and quantitative performance of a complex circuit. Accordingly, this work has set the stage for the predictive design of transcriptional programs of greater complexity.

Human Naa50 Shows Serotonin N-Acetyltransferase Activity, and Its Overexpression Enhances Melatonin Biosynthesis, Resulting in Osmotic Stress Tolerance in Rice.

A new clade of serotonin N-acetyltransferase (SNAT), the penultimate enzyme in the melatonin biosynthetic pathway, has been reported in the archaeon Thermoplasma volcanium. The closest homolog of archaea SNAT in human was an N-alpha-acetyltransferase50 (Naa50). To determine whether human Naa50 (hNaa50) shows SNAT enzyme activity, we chemically synthesized and expressed the hNaa50 gene in Escherichia coli, followed by Ni2+ affinity purification. Purified recombinant hNaa50 showed SNAT activity (Km and Vmax values of 986 µM and 1800 pmol/min/mg protein, respectively). To assess its in vivo function, hNaa50 was overexpressed in rice (hNaa50-OE). The transgenic rice plants produced more melatonin than nontransgenic wild-type rice, indicating that hNaa50 is functionally coupled with melatonin biosynthesis. Due to its overproduction of melatonin, hNaa50-OE had a higher tolerance against osmotic stress than the wild type. Enhanced expression of the chaperone genes BIP1 and CNX in hNaa50-OE plants was responsible for the increased tolerance. It is concluded that hNaa50 harbors serotonin N-acetyltransferase enzyme activity in addition to its initial N-alpha-acetyltransferase, suggesting the bifunctionality of the hNaa50 enzyme toward serotonin and protein substrates. Consequently, ectopic overexpression of hNaa50 in rice enhanced melatonin synthesis, indicating that hNaa50 is in fact involved in melatonin biosynthesis.

A double-strain TM (gp45) polypeptide antigen and its application in the serodiagnosis of equine infectious anemia.

Lentiviruses, including equine infectious anemia virus (EIAV), are considered viral quasispecies because of their intrinsic genetic, structural and phenotypic variability. Immunoenzymatic tests (ELISA) for EIAV reported in the literature were obtained mainly by using the capsid protein p26, which is derived almost exclusively from a single strain (Wyoming), and do not reflect the great potential epitopic variability of the EIAV quasispecies. In this investigation, the GenBank database was exploited in a systematic approach to design a set of representative protein antigens useful for EIAV serodiagnosis. The main bioinformatic tools used were clustering, molecular modelling, epitope predictions and aggregative/ solubility predictions. This approach led to the design of two antigenic proteins, i.e. a full sequence p26 capsid protein and a doublestrain polypeptide derived from the gp45 transmembrane protein fused to Maltose Binding Protein (MBP) that were expressed by recombinant DNA technology starting from synthetic genes, and analyzed by circular dichroism (CD) spectroscopy. Both proteins were used in an indirect ELISA test that can address some of the high variability of EIAV. The novel addition of the gp45 double-strain antigen contributed to enhance the diagnostic sensitivity and could be also useful for immunoblotting application.

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

BACKGROUND: Owing 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. RESULTS: Here, 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. CONCLUSION: The 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.

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.

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.

STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells.

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.

A genetic mammalian proportional-integral feedback control circuit for robust and precise gene regulation.

The processes that keep a cell alive are constantly challenged by unpredictable changes in its environment. Cells manage to counteract these changes by employing sophisticated regulatory strategies that maintain a steady internal milieu. Recently, the antithetic integral feedback motif has been demonstrated to be a minimal and universal biological regulatory strategy that can guarantee robust perfect adaptation for noisy gene regulatory networks in Escherichia coli. Here, we present a realization of the antithetic integral feedback motif in a synthetic gene circuit in mammalian cells. We show that the motif robustly maintains the expression of a synthetic transcription factor at tunable levels even when it is perturbed by increased degradation or its interaction network structure is perturbed by a negative feedback loop with an RNA-binding protein. We further demonstrate an improved regulatory strategy by augmenting the antithetic integral motif with additional negative feedback to realize antithetic proportional-integral control. We show that this motif produces robust perfect adaptation while also reducing the variance of the regulated synthetic transcription factor. We demonstrate that the integral and proportional-integral feedback motifs can mitigate the impact of gene expression burden, and we computationally explore their use in cell therapy. We believe that the engineering of precise and robust perfect adaptation will enable substantial advances in industrial biotechnology and cell-based therapeutics.

Development and evaluation of a qPCR detection method for citrinin in Liupao tea.

Penicillium is universal in dark tea, and Penicillium citrinum can produce a kidney toxin called citrinin (CIT). Determining CIT is difficult because of the complexity of the dark tea substrate and the diversity of CIT-producing fungi. Therefore, this study established a real-time PCR (qPCR) detection method for CIT-related synthetic genes (ctnD, orf1, ctnA, pksCT, orf5, orf7, and ctnG) in Liupao tea and determined the content of CIT in samples at different production stages and the toxin-producing abilities of fungi (Aspergillus oryzae, etc.) in Liupao tea. CIT was found in all samples during the pile-fermentation process of Liupao tea, and CIT was detected in two samples during the aging process. The established method demonstrated good sensitivity and specificity in detecting CIT-related synthetic genes. The reaction efficiency was within the preferred range of 100 ± 10%. CIT was not detected or was below the detection limit when the Ct value of one or more related synthetic genes was greater than 33.5. Therefore, the established qPCR method can effectively predict the production of CIT in Liupao tea, and it is applicable to the judgment of whether fungi produce CIT.

Generation of the salicylic acid deficient Arabidopsis via a synthetic salicylic acid hydroxylase expression cassette.

BACKGROUND: Salicylic acid (SA) is one of the plant hormones, which plays crucial roles in signaling transduction in plant growth, disease resistance, and leaf senescence. Arabidopsis (Arabidopsis thaliana) SA 3-hydroxylase (S3H) and 5-hydroxylase (S5H) are key enzymes which maintain SA homeostasis by catalyzing SA to 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA, respectively. RESULTS: SA deficient transgenic Arabidopsis lines were generated by introducing two binary vectors S5Hpro::EGFP-S3H and 35Spro::EGFP-S3H respectively, in which the expression of S3H is under the control of the S5H promoter or CaMV 35S promoter. Compared with the constitutive expression of S3H gene under the control of 35S promoter, the S3H gene under the native S5H promoter is activated by endogenous SA and results in a dynamic control of SA catabolism in a feedback mode. The SA accumulation, growth, leaf senescence, and pathogen resistance of the S5Hpro::GFP-S3H transgenic plants were investigated in parallel with NahG transgenic plants. The SA levels in the S5Hpro::EGFP-S3H transgenic plants were similar to or slightly lower than those of NahG transgenic Arabidopsis and resulted in SA deficient phenotypes. The low-SA trait of the S5Hpro::EGFP-S3H transgenic lines was inherited stably in the later generations. CONCLUSIONS: Compared with NahG transgenic lines producing by-product catechol, S5Hpro::EGFP-S3H transgenic lines reduce SA levels by converting SA to a native product 2,3-DHBA for catabolism. Together, we provide new SA-deficient germplasms for the investigations of SA signaling in plant development, leaf senescence, and disease resistance.