Field Of Synthetic Biology Research Articles

Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs

Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

Building biosecurity for synthetic biology.

The fast-paced field of synthetic biology is fundamentally changing the global biosecurity framework. Current biosecurity regulations and strategies are based on previous governance paradigms for pathogen-oriented security, recombinant DNA research, and broader concerns related to genetically modified organisms (GMOs). Many scholarly discussions and biosecurity practitioners are therefore concerned that synthetic biology outpaces established biosafety and biosecurity measures to prevent deliberate and malicious or inadvertent and accidental misuse of synthetic biology's processes or products. This commentary proposes three strategies to improve biosecurity: Security must be treated as an investment in the future applicability of the technology; social scientists and policy makers should be engaged early in technology development and forecasting; and coordination among global stakeholders is necessary to ensure acceptable levels of risk.

Bringing Light into Cell-Free Expression.

Cell-free systems, as part of the synthetic biology field, have become a critical platform in biological studies. However, there is a lack of research into developing a switch for a dynamical control of the transcriptional and translational process. The optogenetic tool has been widely proven as an ideal control switch for protein synthesis due to its nontoxicity and excellent time-space conversion. Hence, in this study, a blue light-regulated two-component system named YF1/FixJ was incorporated into an Escherichia coli-based cell-free system to control protein synthesis. The corresponding cell-free system successfully achieved a 5-fold dynamic protein expression by blue light repression and 3-fold dynamic expression by blue light activation. With the aim of expanding the applications of cell-free synthetic biology, the cell-free blue light-sensing system was used to perform imaging, light-controlled antibody synthesis, and light-triggered artificial cell assembly. This study can provide a guide for further research into the field of cell-free optical sensing. Moreover, it will also promote the development of cell-free synthetic biology and optogenetics through applying the cell-free optical sensing system to synthetic biology education, biopharmaceutical research, and artificial cell construction.

A high-resolution map of bacteriophage ϕX174 transcription

Bacteriophage ϕX174 is a model virus for studies across the fields of structural biology, genetics, gut microbiomics, and synthetic biology, but did not have a high-resolution transcriptome until this work. In this study we used next-generation sequencing to measure the RNA produced from ϕX174 while infecting its host E. coli C. We broadly confirm the past transcriptome model while revealing several interesting deviations from previous knowledge. Additionally, we measure the strength of canonical ϕX174 promoters and terminators and discover both a putative new promoter that may be activated by heat shock sigma factors, as well as rediscover a controversial Rho-dependent terminator. We also provide evidence for the first antisense transcription observed in the Microviridae, identify two promoters that may be involved in generating this transcriptional activity, and discuss possible reasons why this RNA may be produced.

Structural Basis for Broad Substrate Selectivity of Alcohol Dehydrogenase YjgB from Escherichia coli.

In metabolic engineering and synthetic biology fields, there have been efforts to produce variable bioalcohol fuels, such as isobutanol and 2-phenylethanol, in order to meet industrial demands. YjgB is an aldehyde dehydrogenase from Escherichia coli that shows nicotinamide adenine dinucleotide phosphate (NADP)-dependent broad selectivity for aldehyde derivatives with an aromatic ring or small aliphatic chain. This could contribute to the design of industrial synthetic pathways. We determined the crystal structures of YjgB for both its apo-form and NADP-complexed form at resolutions of 1.55 and 2.00 Å, respectively, in order to understand the mechanism of broad substrate selectivity. The hydrophobic pocket of the active site and the nicotinamide ring of NADP(H) are both involved in conferring its broad specificity toward aldehyde substrates. In addition, based on docking-simulation data, we inferred that π–π stacking between substrates and aromatic side chains might play a crucial role in recognizing substrates. Our structural analysis of YjgB might provide insights into establishing frameworks to understand its broad substrate specificity and develop engineered enzymes for industrial biofuel synthesis.

State-based supervisory control with restrictions on the supervisor realization

In this paper, we formalize and solve a state-based Supervisory Control problem with restrictions on the supervisor realization that have not been tackled by the Supervisory Control Theory (SCT) community so far. This problem was derived from the application of SCT to intervene in the dynamics of gene regulatory networks, a relevant problem in the fields of Systems and Synthetic Biology. In our framework, a plant, whose states x are represented by Boolean strings, must be driven from an initial state to a target one, by means of m state-feedback control laws ui = hi(x). The Boolean functions hi, though, cannot be freely chosen, but must rather belong to a (possibly strict) subset R of all the Boolean functions on x.

Intuitive biology, moral reasoning, and engineering life: Essentialist thinking and moral purity concerns shape risk assessments of synthetic biology technologies

The field of synthetic biology heralds a new era in our relationship with nature, as organisms are engineered to meet human goals. But little attention has been paid to potential cognitive constraints on reasoning about such technologies. Across four studies with American adults (N = 649), the present research investigates the proposal that essentialist reasoning and moral purity concerns conspire to shape risk assessments of engineered organisms. Moral purity concerns but not moral harm concerns predict moral wrongness judgments of adding a foreign gene to a plant (Studies 1, 2 & 4), as well as assessments of risk (Studies 1 and 2), and risk of harm from eating (Study 4). Adding a gene from a taxonomically distant organism is considered more morally wrong (Studies 2, 3 and 4), more risky (Studies 2 & 3), and more risky to eat (Study 4), than adding either a gene from a similar organism or a new-to-nature gene. Assessments of the risk of gene spread follow a different pattern, with the new-to-nature gene considered safest (Study 4). The findings support the proposal that gene change is reasoned about as essence change that threatens notions of moral purity, with direct implications for certain types of risk perceptions (eating), but not others (gene spread). The findings elucidate cognitive constraints on risk perceptions of synthetic biology, shed fresh light on essentialist and moral reasoning in a novel biological context, and demonstrate the need to differentiate between both risk context and risk type in cognitive accounts of risk perception.

Synthetic Biology Approaches to Engineer Saccharomyces cerevisiae towards the Industrial Production of Valuable Polyphenolic Compounds.

Polyphenols are plant secondary metabolites with diverse biological and potential therapeutic activities such as antioxidant, anti-inflammatory and anticancer, among others. However, their extraction from the native plants is not enough to satisfy the increasing demand for this type of compounds. The development of microbial cell factories to effectively produce polyphenols may represent the most attractive solution to overcome this limitation and produce high amounts of these bioactive molecules. With the advances in the synthetic biology field, the development of efficient microbial cell factories has become easier, largely due to the development of the molecular biology techniques and by the identification of novel isoenzymes in plants or simpler organisms to construct the heterologous pathways. Furthermore, efforts have been made to make the process more profitable through improvements in the host chassis. In this review, advances in the production of polyphenols by genetically engineered Saccharomyces cerevisiae as well as by synthetic biology and metabolic engineering approaches to improve the production of these compounds at industrial settings are discussed.

Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems.

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5′ vs. 3′ untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.

Intrinsic limitations in mainstream methods of identifying network motifs in biology

BackgroundNetwork motifs are connectivity structures that occur with significantly higher frequency than chance, and are thought to play important roles in complex biological networks, for example in gene regulation, interactomes, and metabolomes. Network motifs may also become pivotal in the rational design and engineering of complex biological systems underpinning the field of synthetic biology. Distinguishing true motifs from arbitrary substructures, however, remains a challenge.ResultsHere we demonstrate both theoretically and empirically that implicit assumptions present in mainstream methods for motif identification do not necessarily hold, with the ramification that motif studies using these mainstream methods are less able to effectively differentiate between spurious results and events of true statistical significance than is often presented. We show that these difficulties cannot be overcome without revising the methods of statistical analysis used to identify motifs.ConclusionsPresent-day methods for the discovery of network motifs, and, indeed, even the methods for defining what they are, are critically reliant on a set of incorrect assumptions, casting a doubt on the scientific validity of motif-driven discoveries. The implications of these findings are therefore far-reaching across diverse areas of biology.

Beyond natural: synthetic expansions of botanical form and function.

SummaryPowered by developments that enabled genome‐scale investigations, systems biology emerged as a field aiming to understand how phenotypes emerge from network functions. These advances fuelled a new engineering discipline focussed on synthetic reconstructions of complex biological systems with the goal of predictable rational design and control. Initially, progress in the nascent field of synthetic biology was slow due to the ad hoc nature of molecular biology methods such as cloning. The application of engineering principles such as standardisation, together with several key technical advances, enabled a revolution in the speed and accuracy of genetic manipulation. Combined with mathematical and statistical modelling, this has improved the predictability of engineering biological systems of which nonlinearity and stochasticity are intrinsic features leading to remarkable achievements in biotechnology as well as novel insights into biological function. In the past decade, there has been slow but steady progress in establishing foundations for synthetic biology in plant systems. Recently, this has enabled model‐informed rational design to be successfully applied to the engineering of plant gene regulation and metabolism. Synthetic biology is now poised to transform the potential of plant biotechnology. However, reaching full potential will require conscious adjustments to the skillsets and mind sets of plant scientists.

Expanding the Chemogenetic Toolbox by Circular Permutation

To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12–rapamycin binding domain (cpFRB). By permuting the topology of the four helices within FRB, we have created cpFRB–FKBP pairs that respond to ligand with varying activation kinetics and dynamics. The cpRAPID system enables chemical-controllable subcellular redistribution of proteins, as well as inducible transcriptional activation when coupled with the CRISPR activation (CRISPRa) technology to induce a GFP reporter and endogenous gene expression. We have further demonstrated the use of cpRAPID to generate chemically switchable split nanobody (designated Chessbody) for ligand-gated antigen recognition in living cells. Collectively, the circular permutation approach offers a powerful means for diversifying the chemogenetics toolbox to benefit the burgeoning synthetic biology field.

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles.

Light‐driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS‐g‐PEO and diblock copolymer PBd‐PEO. Activity of more than 90 % in lipid/polymer‐based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long‐term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer‐based hybrids.

Social license and synthetic biology: the trouble with mining terms

ABSTRACT In the wake of controversies over first-generation biotechnologies, the growing field of synthetic biology appears cognizant of the need to attend to the social, political, cultural, and ethical dimensions of innovation. Public engagement has emerged as an important means for attending to these dimensions. Here, we call attention to the problematic nature of one paradigm being drawn upon to conceptualize this public engagement for synthetic biology: social license to operate (SLO). After reviewing SLO’s emergence in the resource extraction context and the existing critiques of SLO, we examine its current use in the synthetic biology literature. We argue that an SLO-derived model of engagement is especially inadequate for synthetic biology due to unique challenges posed by synthetic biology and the limited conception of engagement provided by SLO. We conclude by discussing alternative public engagement paradigms and examples better suited to inform synthetic biology governance.

Noise and Synthetic Biology: How to Deal with Stochasticity?

This paper explores the functional role of noise in synthetic biology and its relation to the concept of randomness. Ongoing developments in the field of synthetic biology are pursuing the re-organisation and control of biological components to make functional devices. This paper addresses the distinction between noise and randomness in reference to the functional relationships that each may play in the evolution of living and/or synthetic systems. The differentiation between noise and randomness in its constructive role, that is, between noise as a perturbation in routine behaviours and noise as a source of variability that cells may exploit, indicates the need for a clarification and rectification (whenever necessary) of the conflicting uses of the notion of noise in the studies of the so-called noise biology.

Silk Protein Solution: A Natural Example of Sticky Reptation.

Silk is one of the most intriguing examples of biomolecular self-assembly, yet little is understood of molecular mechanisms behind the flow behavior generating these complex high-performance fibers. This work applies the polymer physics of entangled solution rheology to present a first microphysical understanding of silk in the linear viscoelastic regime. We show that silk solutions can be approximated as reptating polymers with “sticky” calcium bridges whose strength can be controlled through the potassium concentration. This approach provides a new window into critical microstructural parameters, in particular identifying the mechanism by which potassium and calcium ions are recruited as a powerful viscosity control in silk. Our model constitutes a viable starting point to understand not only the “flow-induced self-assembly” of silk fibers but also a broader range of phenomena in the emergent field of material-focused synthetic biology.

An expanded library of orthogonal split inteins enables modular multi-peptide assemblies

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.

Quorum Sensing System Used as a Tool in Metabolic Engineering.

Quorum sensing (QS) is a ubiquitous cell-cell communication mechanism in microbes that coordinates population-level cell behaviors, such as biofilm production, virulence, swarming motility, and bacterial persistence. Efforts to engineer QS systems to take part in metabolic network regulation represent a promising strategy for synthetic biology and pathway engineering. Recently, design, construction, and implementation of QS circuits for programmed control of bacterial phenotypes and metabolic pathways have gained much attention, but have not been reviewed recently. In this article, the architectural organizations and genetic contributions of the naturally occurring QS components to understand the mechanisms are summarized. Then, the most recent progress in application of QS toolkits to develop synthetic networks for novel cell behaviors creation and metabolic pathway engineering is highlighted. The current challenges in large-scale application of these QS circuits in synthetic biology and metabolic engineering fields are discussed and future perspectives for further engineering efforts are provided.

The Q-System as a Synthetic Transcriptional Regulator in Plants.

A primary focus of the rapidly growing field of plant synthetic biology is to develop technologies to precisely regulate gene expression and engineer complex genetic circuits into plant chassis. At present, there are few orthogonal tools available for effectively controlling gene expression in plants, with most researchers instead using a limited set of viral elements or truncated native promoters. A powerful repressible-and engineerable-binary system that has been repurposed in a variety of eukaryotic systems is the Q-system from Neurospora crassa. Here, we demonstrate the functionality of the Q-system in plants through transient expression in soybean (Glycine max) protoplasts and agroinfiltration in Nicotiana benthamiana leaves. Further, using functional variants of the QF transcriptional activator, it was possible to modulate the expression of reporter genes and to fully suppress the system through expression of the QS repressor. As a potential application for plant-based biosensors (phytosensors), we demonstrated the ability of the Q-system to amplify the signal from a weak promoter, enabling remote detection of a fluorescent reporter that was previously undetectable. In addition, we demonstrated that it was possible to coordinate the expression of multiple genes through the expression of a single QF activator. Based on the results from this study, the Q-system represents a powerful orthogonal tool for precise control of gene expression in plants, with envisioned applications in metabolic engineering, phytosensors, and biotic and abiotic stress tolerance.

A Practicable Method of Tuning the Noise Intensity at Protein Level.

The expression of genes is inevitably subject to intracellular noise. Noise, for some regulatory networks, is constructive but detrimental to many others. The intensity of the noise is a determinant factor and the method of tuning it is of great value. In this study, we illustrated that the transcriptional delay in an incoherent feedforward loop (FFL) grants the target protein modulation the intensity of noise. Remarkably, for a wide range, the coefficient of variation (COV) of the target protein appeared to be about linear to the time span of the transcriptional delay. Without a noise-buffering method, the COV of the target protein is 0.455. While applying incoherent FFL, the COV reduced to 0.236. Then, it changed from 0.236 to 0.630 as the transcriptional delay raised from 0 to 1000 seconds. If we further increased the delay out of the linear range, the COV finally reached 0.779. In addition, we incorporated the distribution of the transcriptional delay in the delay stochastic simulation algorithm. This distribution is based on the experimental observation in the literature. The outcome suggested that the distributed delay slightly improved the ability of tuning noise. In conclusion, we demonstrated a noise-tuning method that altered only the intensity of noise without changing the deterministic steady-state behaviors. It is ready to be applied to various systems in the field of synthetic biology.