Field Of Synthetic Biology Research Articles

The Fold-Illuminator: A low-cost, portable, and disposable incubator-illuminator device

Fluorescent reporters have revolutionized modern applications in the fields of molecular and synthetic biology, enabling applications ranging from education to point-of-care diagnostics. Past advancements in these fields have primarily focused on improving reaction conditions, the development of new applications, and the broad dissemination of these technologies. However, field and classroom-based applications have remained limited in part due to the nature of fluorescent signal detection, which often requires the use of costly lab equipment to observe and quantify fluorescence readouts. Users without access to laboratory equipment rely on qualitative assessments of fluorescence, a process that remains highly variable from user-to-user even within the same classroom. To overcome this challenge, we have developed a foldable illuminator and incubator device to support field-applications of synthetic biology-based biosensors for education and diagnostics. The Fold-Illuminator is an affordable, portable, and recyclable device that allows for the visible detection of fluorescent biomolecules. The Fold-Illuminator's design allows for assembly in under 10 min, a user can then utilize the optional heating element to incubate biochemical reactions and visualize fluorescence outputs in a defined and light-controlled environment. Interchangeable LED strips and light-filtering screens provide modularity to pair with the fluorescence wavelengths of interest. The user can then unfold the device for convenient storage, transport, or even recycling. The cost for the Fold-Illuminator is $5.58 USD and is compatible with an optional heating element for an additional $3.98 cost, with potential for further reductions in cost for larger quantities. Open-source templates for cutting device parts from paper stock are provided for both printing and cutting by hand; cutting can also be achieved with consumer-grade smart cutting machines such as the Cricut®. Combined with the broad applications of fluorescent reporters, the Fold-Illuminator has the potential to improve access to fluorescence visualization and quantification for new users as well as emerging field applications.

A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)

Current molecular cloning strategies generally lack inter-compatibility, are not strictly modular, or are not applicable to engineer multi-gene expression vectors for transient and stable integration. A standardized molecular cloning platform would advance research, for example, by promoting exchange of vectors between groups. Here, we present a versatile plasmid architecture for mammalian synthetic biology, which we designate VAMSyB, consisting of a three-tier vector family. Tier-1 is designed for easy engineering of fusion constructs, as well as easy swapping of genes and modules to tune the functionality of the vector. Tier-2 is designed for transient multi-gene expression, and is constructed by directly transferring the engineered expression cassettes from tier-1 vectors. Tier-3 enables stable integration into a mammalian host cell through viral transduction, transposons, or homology-directed recombination via CRISPR. This VAMSyB architecture is expected to have broad applicability in the field of mammalian synthetic biology. The VAMSyB collection of plasmids will be available through Addgene.

Designing Biological Circuits: Synthetic Biology Within the Operon Model and Beyond.

In 1961, Jacob and Monod proposed the operon model of gene regulation. At the model's core was the modular assembly of regulators, operators, and structural genes. To illustrate the composability of these elements, Jacob and Monod linked phenotypic diversity to the architectures of regulatory circuits. In this review, we examine how the circuit blueprints imagined by Jacob and Monod laid the foundation for the first synthetic gene networks that launched the field of synthetic biology in 2000. We discuss the influences of the operon model and its broader theoretical framework on the first generation of synthetic biological circuits, which were predominantly transcriptional and posttranscriptional circuits. We also describe how recent advances in molecular biology beyond the operon model-namely, programmable DNA- and RNA-binding molecules as well as models of epigenetic and posttranslational regulation-are expanding the synthetic biology toolkit and enabling the design of more complex biological circuits.

Improvement of Biomedical Structural Polymers by Synthetic Biology Methods

Materials that have a biological origin find many applications in both Biomedicine and other Sciences. However, the properties of these materials are difficult to change, since natural biosynthetic mechanisms are difficult to explain, simulate, or adjust. Thus, many materials of biological origin are isolated from natural tissues or their substitutes are produced recombinantly, and then modified in the process of experimental application.
 A major shift in this paradigm is caused by the emerging field of synthetic biology, which introduces innovations in the "tool kit" of methods for tuning biomolecules and biosynthetic mechanisms. Relative to materials, this led to higher product titers due to reprogrammed natural biosynthesis and allowed the development of new materials by combining the desired domains. Here we can focus on recent applications of synthetic biology to bio-derived ribosomal and non-ribosomal polymer materials for biomedical applications. It is also interesting to describe modern methods that will affect the production and design of biomaterials in the near future. Continuous innovation at the intersection of synthetic biology and materials science promises to usher in a new era of biomaterial design and synthesis.

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology.

Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

Invest in Canadian Synthetic Biology to Meet Commitments to Sustainable Development and Support Economic Recovery

Canadian post-COVID-19 economic recovery efforts have been framed around values aligned with Canada's commitments to the United Nations Sustainable Development Goals (SDGs), primarily concerning environmental sustainability. The field of synthetic biology (synbio) offers many innovative ways to achieve these goals while growing the economy. Here, we discuss the opportunity for Canada to become a leader in clean technology applications of synbio. Investments in synthetic biology, which has traditionally been underfunded compared to other countries, will have beneficial impacts on the environment while driving Canada's post-pandemic economic recovery.

Microbial engineering to produce fatty alcohols and alkanes.

Owing to their high energy density and composition, fatty acid-derived chemicals possess a wide range of applications such as biofuels, biomaterials, and other biochemical, and as a consequence, the global annual demand for products has surpassed 2 million tons. With the exhausting petroleum reservoirs and emerging environmental concerns on using petroleum feedstock, it has become indispensable to shift to a renewable-based industry. With the advancement in the field of synthetic biology and metabolic engineering, the use of microbes as factories for the production of fatty acid-derived chemicals is becoming a promising alternative approach for the production of these derivatives. Numerous metabolic approaches have been developed for conditioning the microbes to improve existing or develop new methodologies capable of efficient oleochemical production. However, there still exist several limitations that need to be addressed for the commercial viability of the microbial cell factory production. Though substantial advancement has been made toward successfully producing these fatty acids derived chemicals, a considerable amount of work needs to be done for improving the titers. In the present review, we aim to address the roadblocks impeding the heterologous production, the engineering pathway strategies implemented across the range of microbes in a detailed manner, and the commercial readiness of these molecules of immense application.

Genetically Encodable Scaffolds for Optimizing Enzyme Function.

Enzyme engineering is an indispensable tool in the field of synthetic biology, where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial production of chemicals, biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biology methodologies, to complement the purposeful deployment of enzymes. Current molecular tools for constructing these synthetic enzyme-scaffold systems are also highlighted.

Recent developments in microfluidic synthesis of artificial cell-like polymersomes and liposomes for functional bioreactors.

Recent advances in droplet microfluidics have led to the fabrication of versatile vesicles with a structure that mimics the cellular membrane. These artificial cell-like vesicles including polymersomes and liposomes effectively enclose an aqueous core with well-defined size and composition from the surrounding environment to implement various biological reactions, serving as a diverse functional reactor. The advantage of realizing various biological phenomena within a compartment separated by a membrane that resembles a natural cell membrane is actively explored in the fields of synthetic biology as well as biomedical applications including drug delivery, biosensors, and bioreactors, to name a few. In this Perspective, we first summarize various methods utilized in producing these polymersomes and liposomes. Moreover, we will highlight some of the recent advances in the design of these artificial cell-like vesicles for functional bioreactors and discuss the current issues and future perspectives.

Microfluidic approaches for synthetic gene circuits’ construction and analysis

BackgroundMicrofluidic systems have advantages such as a high throughput, small reaction volume, and precise control of the cellular position and environment. These advantages have allowed microfluidics to be widely used in several fields of synthetic biology in recent years.ResultsIn this article, we reviewed the microfluidic‐based methods for synthetic biology from two aspects: the construction of synthetic gene circuits and the analysis of synthetic gene systems. We used some examples to illuminate the progresses and challenges in the steps of synthetic gene circuits construction and approaches of gene expression analysis with microfluidic systems.ConclusionComparing to traditional methods, microfluidic tools promise great advantages in the synthetic genetic circuit building and analysis process. Moreover, new microfluidic systems together with the mathematical modeling of synthetic circuits or consortiums are desirable to perform complex genetic circuit construction and understand the natural gene regulation in cells and population interactions better.

합성생물학 전수조사, 모니터링 및 평가체계 수립에 대한 국제적 논의 동향 및 국내 대응 방안 연구

합성생물학(synthetic biology)은 현재 생물다양성협약과 그 부속의정서인 카르타헤나의정서와 나고야의정서에서 가장 주목받고 있는 교차영역의제(cross-cutting issues) 중의 하나이다. 국제협약에서 합성생물학이 중요한 주제로 논의될 수 있는 법적 근거는 생물다양성협약의 전문과 카르타헤나의정서 제10(6)조에 명시된 사전주의 원칙(precautionary principle)이다. 사전주의 원칙에 따라 합성생물학의 잠재적 부작용에 대한 과학적 근거는 부족할지라도 그 위험성에 대한 우려의 제기로 합성생물학 규율을 논의할 수 있다.<BR> 2021년에 개최될 제15차 생물다양성협약 당사국총회에서는, 합성생물학에 대한 제도적 규율의 일환으로, 합성생물학 기술개발에 대한 전수조사, 모니터링 및 평가(horizon scanning, monitoring and assessment) 절차 수립 및 이를 지원할 다분야기술전문가그룹(multidisciplinary technical expert group) 설치에 대한 합의를 앞두고 있다. 이는 제14차 생물다양성협약 당사국총회에서 합성생물학의 잠재적 긍정적 영향과 잠재적 부정적 영향에 관한 새로운 정보를 검토하기 위해서 가장 최신 기술개발에 대한 광범위하고 정기적인 전수조사, 모니터링 및 평가가 필요하다는데 당사국들이 동의한 결과이다.<BR> 한편 국제자연연맹(IUCN)과 같은 국제기구들과 선진국들 또한 합성생물학의 잠재적 부작용에 대한 연구 및 대응 방안을 서두르고 있다. 최근 미국에서는 합성유기체의 잠재적인 부정적 영향을 현재의 규제 시스템으로 규제하는 데는 한계가 있으며, 앞으로 매우 많은 수의 합성유기체가 만들어질 것이고, 이것이 환경에 막대한 영향을 미칠 가능성을 제기하였다. 영국과 독일도 합성유기체는 자연을 기반으로 발생한 생물들과 전혀 다르므로 합성유기체만을 규제하는 새로운 규제제도 수립의 필요성을 제기하였다.<BR> 이처럼 합성생물학의 잠재적 부작용에 대한 우려가 증가함에 따라 선진국과 국제기구는 대응방안 마련을 서두르고 있다. 그러나 우리나라는 기초 조사는 물론 기존 유전자변형생물체와 차원이 다른 새로운 모니터링과 평가체계 수립이 필요하다는 인식조차 하지 못하고 있는 실정이다. 이제는 우리나라도 합성생물학 적용 생성물이 인간, 환경에 끼칠 잠재적 부작용에 대해 주목할 시기가 되었다.<BR> 이에 본 연구는 먼저 국제협약에서 합성생물학 최신 기술발전에 대한 전수조사, 모니터링 및 평가체계가 논의되기 시작한 배경 및 과정, 그리고 지금까지 국제적으로 합의된 사항에 대해서 살펴보고자 한다. 다음으로 합성생물학 전수조사, 모니터링 및 평가체계 수립을 두고 제시되고 있는 주요 내용을 살펴보고자 한다. 끝으로 이러한 연구를 바탕으로 제도적 측면에서 국내 대응 방안 및 정책적 과제를 제시하고자 한다.

Prokaryote playhouse: A low-cost, laser-cut acrylic incubator for optogenetic bacterial culture

Bacterial photography is a printing technique that replaces conventional photochemistry with a living film of engineered Escherichia coli. Many biology teaching labs have adopted monochrome bacterial photography because it offers a captivating playground for illustrating central concepts and lab techniques in biological engineering, particularly in the fields of synthetic biology and optogenetics. Recent improvements have increased the number of color channels from one to three. A key practical challenge in three-color printing is to expose a Petri dish loaded with engineered bacteria to a trichromatic image while maintaining it at 37 °C. Prokaryote Playhouse is a compact, inexpensive, open-source, benchtop incubator for light-sensitive bacterial cultures that makes bacterial photography and similar bacterial optogenetic methods more accessible to teaching labs, makerspaces, and research labs. The system includes a laser-cut, light-tight enclosure; digital thermostat; heated sample shelf; single-board computer; and miniature projector. We built a fleet of Prokaryote Playhouses that students have used to produce hundreds of bacterial photographs in a wide range of educational experiences, ranging from a four-hour introduction to synthetic biology and wet lab techniques to a six-week exploratory class for first-year students at MIT.

Research progress and the biotechnological applications of multienzyme complex.

The multienzyme complex system has become a research focus in synthetic biology due to its highly efficient overall catalytic ability and has been applied to various fields. Multienzyme complexes are formed by cascading complexes, which are multiple functionally related enzymes that continuously and efficiently catalyze the production of substrates. Compared with current mainstream microbial cell catalytic systems, in vitro multienzyme molecular machines have many advantages, such as fewer side reactions, a high product yield, a fast reaction speed, easy product separation, a tolerable toxic environment, and robust system operability, showing increasing competitiveness in the field of biomanufacturing. In this review, the research progress of multienzyme complexes in nature and multienzyme cascades in vivo or in vitro will be introduced, and the discovered enzyme cascades concerning scaffolding proteins will also be discussed. This review is expected to provide a more theoretical basis for the modification of multienzyme complexes and broaden their application in the field of synthetic biology. KEY POINTS: • The cascade reactions of some natural multienzyme complexes are reviewed. • The main approaches of constructing artificial multienzyme complexes are summarized. • The structure and application of cellulosomes are discussed and prospected.

Redesigning the Genetic Polymers of Life.

Genomes can be viewed as constantly updated memory systems where information propagated in cells is refined over time by natural selection. This process, commonly known as heredity and evolution, has been the sole domain of DNA since the origin of prokaryotes. Now, some 3.5 billion years later, the pendulum of discovery has swung in a new direction, with carefully trained practitioners enabling the replication and evolution of "xeno-nucleic acids" or "XNAs"-synthetic genetic polymers in which the natural sugar found in DNA and RNA has been replaced with a different type of sugar moiety. XNAs have attracted significant attention as new polymers for synthetic biology, biotechnology, and medicine because of their unique physicochemical properties that may include increased biological stability, enhanced chemical stability, altered helical geometry, or even elevated thermodynamics of Watson-Crick base pairing.This Account describes our contribution to the field of synthetic biology, where chemical synthesis and polymerase engineering have allowed my lab and others to extend the concepts of heredity and evolution to synthetic genetic polymers with backbone structures that are distinct from those found in nature. I will begin with a discussion of α-l-threofuranosyl nucleic acid (TNA), a specific type of XNA that was chosen as a model system to represent any XNA system. I will then proceed to discuss advances in organic chemistry that were made to enable the synthesis of gram quantities of TNA phosphoramidites and nucleoside triphosphates, the monomers used for solid-phase and polymerase-mediated TNA synthesis, respectively. Next, I will recount our development of droplet-based optical sorting (DrOPS), a single-cell microfluidic technique that was established to evolve XNA polymerases in the laboratory. This section will conclude with structural insights that have been gained by solving X-ray crystal structures of a laboratory-evolved TNA polymerase and a natural DNA polymerase that functions with general reverse transcriptase activity on XNA templates.The final passage of this Account will examine the role that XNAs have played in synthetic biology by highlighting examples in which engineered polymerases have enabled the evolution of biologically stable affinity reagents (aptamers) and catalysts (XNAzymes) as well as the storage and retrieval of binary information encoded in electronic word and picture file formats. Because these examples provide only a glimpse of what the future may have in store for XNA, I will conclude the Account with my thoughts on how synthetic genetic polymers could help drive new innovations in synthetic biology and molecular medicine.

Giant Vesicles Mimicking Outer Membrane of Gram-Negative Bacterium

Engineering of synthetic cells which mimics the native membrane opens up various platforms for research in membrane biology as well as synthetic biology field. In this work, giant unilamellar vesicles (GUV) mimicking outer membrane of Gram-negative bacterium is engineered by incorporating lipopolysaccharide (LPS) under physiological salt condition. We successfully incorporated LPS in Gram-negative (GN) vesicles by inverse-phase precursor film assembly using gel-assisted technology and confirmed at single-vesicle level using fluorescence detection system. Effective integration of LPS in vesicles is attained in presence of Ca2+ ions at a ratio of LPS: E. coli polar lipid extract (GN extract) (1:6) to construct outer membrane mimicking (OMM) vesicles comparable to the bacterial outer-membrane composition. Quantitative transport rates of antibiotic showed a much slower flux across vesicles with LPS (OMM) compared to GN vesicles further confirming the incorporation of LPS in vesicles and its significance in native bacterial cells. To demonstrate the functionality of engineered OMM vesicles, a model peptide melittin is reconstituted in the membrane of both vesicles and the uptake of small different sized molecules is studied. In GN vesicles, a steady uptake of large molecules is observed in peptide: lipid (1:15) ratio, where in OMM vesicles pore formation and diffusion of molecules occurred at higher peptide: lipid (1:5) ratio confirming intrinsic resistance of OMM vesicles due to presence of LPS. The OMM vesicles constructed here will find immense applications in fundamental understanding of membrane biophysical phenomena.

Synthetic biology in biofilms: Tools, challenges, and opportunities.

The field of synthetic biology seeks to program living cells to perform novel functions with applications ranging from environmental biosensing to smart cell-based therapeutics. Bacteria are an especially attractive chassis organism due to their rapid growth, ease of genetic manipulation, and ability to persist across many environmental niches. Despite significant progress in bacterial synthetic biology, programming bacteria to perform novel functions outside the well-controlled laboratory context remains challenging. In contrast to planktonic laboratory growth, bacteria in nature predominately reside in the context of densely packed communities known as biofilms. While biofilms have historically been considered environmental and biomedical hazards, their physiology and emergent behaviors could be leveraged for synthetic biology to engineer more capable and robust bacteria. Specifically, bacteria within biofilms participate in complex emergent behaviors such as collective organization, cell-to-cell signaling, and division of labor. Understanding and utilizing these properties can enable the effective deployment of engineered bacteria into natural target environments. Toward this goal, this review summarizes the current state of synthetic biology in biofilms by highlighting new molecular tools and remaining biological challenges. Looking to future opportunities, advancing synthetic biology in biofilms will enable the next generation of smart cell-based technologies for use in medicine, biomanufacturing, and environmental remediation.

Standards. The building blocks of complexity

Without standards, the world as we know it would not be possible. International and supra-cultural standards and norms have been a key factor in engineering, as well as in the development of industrial societies. Despite the obvious successes in electronic and mechanical design, other technological areas present difficulties for the application of standards. In the field of biotechnology and synthetic biology – which aims at studying living things from an engineering perspective – standards are desirable, but whether they can be widely adopted remains to be proved.&#x0D; This monograph reviews the sociological and scientific aspects of standardisation and delves into the more problematic facets of universal standardisation, especially in the biological field. Are standards possible in synthetic biology at all? What are the limitations to the universal use of modular and interchangeable parts in a cellular context? Could it be that the biological world resists standardisation, similarly to the field of software engineering, where these attempts have not progressed? And should some kind of standard be applicable in synthetic biology, what qualities might be required in an environment of open science and responsible research and innovation?

Folding RNA-Protein Complex into Designed Nanostructures.

RNA-protein (RNP) complexes are promising biomaterials for the fields of nanotechnology and synthetic biology. Protein-responsive RNA sequences (RNP motifs) can be integrated into various RNAs, such as messenger RNA, short-hairpin RNA, and synthetic RNA nanoobjects for a variety of purposes. Direct observation of RNP interaction in solution at high resolution is important in the design and construction of RNP-mediated nanostructures. Here we describe a method to construct and visualize RNP nanostructures that precisely arrange a target protein on the RNA scaffold with nanometer scale. High-speed AFM (HS-AFM) images of RNP nanostructures show that the folding of RNP complexes of defined sizes can be directly visualized at single RNP resolution in solution.

Composable Rate-Independent Computation in Continuous Chemical Reaction Networks.

Biological regulatory networks depend upon chemical interactions to process information. Engineering such molecular computing systems is a major challenge for synthetic biology and related fields. The chemical reaction network (CRN) model idealizes chemical interactions, allowing rigorous reasoning about the computational power of chemical kinetics. Here we focus on function computation with CRNs, where we think of the initial concentrations of some species as the input and the equilibrium concentration of another species as the output. Specifically, we are concerned with CRNs that are rate-independent (the computation must be correct independent of the reaction rate law) and composable ( f°g can be computed by concatenating the CRNs computing f and g). Rate independence and composability are important engineering desiderata, permitting implementations that violate mass-action kinetics, or even "well-mixedness", and allowing the systematic construction of complex computation via modular design. We show that to construct composable rate-independent CRNs, it is necessary and sufficient to ensure that the output species of a module is not a reactant in any reaction within the module. We then exactly characterize the functions computable by such CRNs as superadditive, positive-continuous, and piecewise rational linear. Thus composability severely limits rate-independent computation unless more sophisticated input/output encodings are used.

CRISPR-Cas systems: Challenges and future prospects.

The advancement gained over the past couple of decades in clustered regularly interspaced short palindromic repeats and CRISPR associated proteins (CRISPR-Cas) systems have revolutionized the field of synthetic biology, therapeutics, diagnostics and metabolic engineering. The technique has enabled the process of genome editing to be very precise, rapid, cost-effective and highly efficient which were the downfalls for the previously debuted zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) technologies. However, despite its great potential, challenges including off-target activity, method of delivery, ethical and regulatory issues still remain unresolved for the CRISPR-Cas systems. In this chapter, we present and point out the obstacles faced in implementation of the CRISPR-Cas system along with its future prospects.