Field Of Synthetic Biology Research Articles

Cell-Free Biosensing Genetic Circuit Coupled with Ribozyme Cleavage Reaction for Rapid and Sensitive Detection of Small Molecules.

Synthetic biological systems have been utilized to develop a wide range of genetic circuits and components that enhance the performance of biosensing systems. Among them, cell-free systems are emerging as important platforms for synthetic biology applications. Genetic circuits play an essential role in cell-free systems, mainly consisting of sensing modules, regulation modules, and signal output modules. Currently, fluorescent proteins and aptamers are commonly used as signal outputs. However, these signal output modes cannot simultaneously achieve faster signal output, more accurate and reliable performance, and signal amplification. Ribozyme is a highly structured and catalytic RNA molecule that can specifically recognize and cut specific substrate sequences. Here, by adopting ribozyme as the signal output, we developed a cell-free biosensing genetic circuit coupled with the ribozyme cleavage reaction, enabling rapid and sensitive detection of small molecules. More importantly, we have also successfully constructed a 3D-printed sensor array and thereby achieved high-throughput analysis of an inhibitory drug. Furthermore, our method will help expand the application range of ribozyme in the field of synthetic biology and also optimize the signal output system of cell-free biosensing, thus promoting the development of cell-free synthetic biology in biomedical research, clinical diagnosis, environmental monitoring, and food inspection.

Simple and rapid detection of tyrosinase activity with the adjustable light scattering properties of CoOOH nanoflakes.

Tyrosinase (TYR), as an important biological enzyme, has been widely used in synthetic biology, medical hairdressing, environmental detection, biological sensors, and other fields. In clinical practice, tyrosinase activity is an important indicator for detecting melanoma. Therefore, the detection of tyrosinase activity is of great importance. Based on the polyphenol oxidase activity of tyrosinase, a simple and rapid detection method was proposed based on the adjustable light scattering properties of cobalt hydroxyl oxide nanoflakes (CoOOH NFs). It was found that the amount and size of CoOOH NFs decreased due to the redox reaction mediated by catechol (CC), resulting in a lower light scattering signal of CoOOH NFs. However, in the presence of tyrosinase, catechol was oxidized to a quinone structure, resulting in the reduced decomposition of CoOOH NFs and recovered light scattering signal, which was developed for the quantitative detection of tyrosinase activity. It was found that in the range of 10-400 U/L, the light scattering intensity wascorrelated linearly with tyrosinase activity, and the limit of detection was 6.71 U/L (3σ/k). To verify the feasibility of the proposed method in clinical samples, thespiked recovery experiments were carried out with human serum samples, which showed recovery rates between 93.0% and 104.6%, suggesting the high accuracy. The proposed assay provides a simple and rapid method for detection of a natural enzyme based on the adjustable light scattering properties of CoOOH nanoflakes, which lays the foundation for the development of various enzyme sensing applications in the future.

Opportunities to accelerate extracellular vesicle research with cell‐free synthetic biology

AbstractExtracellular vesicles (EVs) are lipid‐membrane nanoparticles that are shed or secreted by many different cell types. The EV research community has rapidly expanded in recent years and is leading efforts to deepen our understanding of EV biological functions in human physiology and pathology. These insights are also providing a foundation on which future EV‐based diagnostics and therapeutics are poised to positively impact human health. However, current limitations in our understanding of EV heterogeneity, cargo loading mechanisms and the nascent development of EV metrology are all areas that have been identified as important scientific challenges. The field of synthetic biology is also contending with the challenge of understanding biological complexity as it seeks to combine multidisciplinary scientific knowledge with engineering principles, to build useful and robust biotechnologies in a responsible manner. Within this context, cell‐free systems have emerged as a powerful suite of in vitro biotechnologies that can be employed to interrogate fundamental biological mechanisms, including the study of aspects of EV biogenesis, or to act as a platform technology for medical biosensors and therapeutic biomanufacturing. Cell‐free gene expression (CFE) systems also enable in vitro protein production, including membrane proteins, and could conceivably be exploited to rationally engineer, or manufacture, EVs loaded with bespoke molecular cargoes for use in foundational or translational EV research. Our pilot data herein, also demonstrates the feasibility of cell‐free EV engineering. In this perspective, we discuss the opportunities and challenges for accelerating EV research and healthcare applications with cell‐free synthetic biology.

DIGGER-Bac: prediction of seed regions for high-fidelity construction of synthetic small RNAs in bacteria.

Synthetic small RNAs (sRNAs) are gaining increasing attention in the field of synthetic biology and bioengineering for efficient post-transcriptional regulation of gene expression. However, the optimal design of synthetic sRNAs is challenging because alterations may impair functions or off-target effects can arise. Here, we introduce DIGGER-Bac, a toolbox for Design and Identification of seed regions for Golden Gate assembly and Expression of synthetic sRNAs in Bacteria. The SEEDling tool predicts optimal sRNA seed regions in combination with user-defined sRNA scaffolds for efficient regulation of specified mRNA targets. Results are passed on to the G-GArden tool, which assists with primer design for high-fidelity Golden Gate assembly of the desired synthetic sRNA constructs.

Speed and Correctness Guarantees for Programmable Enthalpy-Neutral DNA Reactions†.

Molecular control circuits embedded within chemical systems to direct molecular events have transformative applications in synthetic biology, medicine, and other fields. However, it is challenging to understand the collective behavior of components due to the combinatorial complexity of possible interactions. Some of the largest engineered molecular systems to date have been constructed using DNA strand displacement reactions, in which signals can be propagated without a net change in base pairs (enthalpy neutral). This flexible and programmable component has been used for constructing molecular logic circuits, smart structures and devices, for systems with complex autonomously generated dynamics, and for diagnostics. Limiting their utility, however, strand displacement systems are susceptible to the spurious release of output in the absence of the proper combination of inputs (leak), as well as reversible unproductive binding (toehold occlusion) and spurious displacement that slow down desired kinetics. We systematize the properties of the simplest enthalpy-neutral strand displacement cascades (logically linear topology), and develop a taxonomy for the desired and undesired properties affecting speed and correctness, and trade-offs between them based on a few fundamental parameters. We also show that enthalpy-neutral linear cascades can be engineered with stronger thermodynamic guarantees to leak than non-enthalpy-neutral designs. We confirm our theoretical analysis with laboratory experiments comparing the properties of different design parameters. Our method of tackling the combinatorial complexity using mathematical proofs can guide the engineering of robust and efficient molecular algorithms.

Measuring Encapsulation Efficiency in Cell-Mimicking Giant Unilamellar Vesicles.

One of the main drivers within the field of bottom-up synthetic biology is to develop artificial chemical machines, perhaps even living systems, that have programmable functionality. Numerous toolkits exist to generate giant unilamellar vesicle-based artificial cells. However, methods able to quantitatively measure their molecular constituents upon formation is an underdeveloped area. We report an artificial cell quality control (AC/QC) protocol using a microfluidic-based single-molecule approach, enabling the absolute quantification of encapsulated biomolecules. While the measured average encapsulation efficiency was 11.4 ± 6.8%, the AC/QC method allowed us to determine encapsulation efficiencies per vesicle, which varied significantly from 2.4 to 41%. We show that it is possible to achieve a desired concentration of biomolecule within each vesicle by commensurate compensation of its concentration in the seed emulsion. However, the variability in encapsulation efficiency suggests caution is necessary when using such vesicles as simplified biological models or standards.

Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR.

Peptides and proteins containing non-canonical amino acids (ncAAs) are a large and important class of biopolymers. They include non-ribosomally synthesised peptides, post-translationally modified proteins, expressed or synthesised proteins containing unnatural amino acids, and peptides and proteins that are chemically modified. Here, we describe a general procedure for generating atomic descriptions required to incorporate ncAAs within popular NMR structure determination software such as CYANA, CNS, Xplor-NIH and ARIA. This procedure is made publicly available via the existing Automated Topology Builder (ATB) server (https://atb.uq.edu.au, last access: 17 February 2023) with all submitted ncAAs stored in a dedicated database. The described procedure also includes a general method for linking of sidechains of amino acids from CYANA templates. To ensure compatibility with other systems, atom names comply with IUPAC guidelines. In addition to describing the workflow, 3D models of complex natural products generated by CYANA are presented, including vancomycin. In order to demonstrate the manner in which the templates for ncAAs generated by the ATB can be used in practice, we use a combination of CYANA and CNS to solve the structure of a synthetic peptide designed to disrupt Alzheimer-related protein-protein interactions. Automating the generation of structural templates for ncAAs will extend the utility of NMR spectroscopy to studies of more complex biomolecules, with applications in the rapidly growing fields of synthetic biology and chemical biology. The procedures we outline can also be used to standardise the creation of structural templates for any amino acid and thus have the potential to impact structural biology more generally.

Transfer of knowledge from model organisms to evolutionarily distant non-model organisms: The coral Pocillopora damicornis membrane signaling receptome.

With the ease of gene sequencing and the technology available to study and manipulate non-model organisms, the extension of the methodological toolbox required to translate our understanding of model organisms to non-model organisms has become an urgent problem. For example, mining of large coral and their symbiont sequence data is a challenge, but also provides an opportunity for understanding functionality and evolution of these and other non-model organisms. Much more information than for any other eukaryotic species is available for humans, especially related to signal transduction and diseases. However, the coral cnidarian host and human have diverged over 700 million years ago and homologies between proteins in the two species are therefore often in the gray zone, or at least often undetectable with traditional BLAST searches. We introduce a two-stage approach to identifying putative coral homologues of human proteins. First, through remote homology detection using Hidden Markov Models, we identify candidate human homologues in the cnidarian genome. However, for many proteins, the human genome alone contains multiple family members with similar or even more divergence in sequence. In the second stage, therefore, we filter the remote homology results based on the functional and structural plausibility of each coral candidate, shortlisting the coral proteins likely to have conserved some of the functions of the human proteins. We demonstrate our approach with a pipeline for mapping membrane receptors in humans to membrane receptors in corals, with specific focus on the stony coral, P. damicornis. More than 1000 human membrane receptors mapped to 335 coral receptors, including 151 G protein coupled receptors (GPCRs). To validate specific sub-families, we chose opsin proteins, representative GPCRs that confer light sensitivity, and Toll-like receptors, representative non-GPCRs, which function in the immune response, and their ability to communicate with microorganisms. Through detailed structure-function analysis of their ligand-binding pockets and downstream signaling cascades, we selected those candidate remote homologues likely to carry out related functions in the corals. This pipeline may prove generally useful for other non-model organisms, such as to support the growing field of synthetic biology.

Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system

BackgroundDespite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming.ResultsIn this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes.ConclusionsThe novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.

Is Public Engagement in Bioengineering and Synthetic Biology Improving Research Outcomes?

Engaging diverse publics on the acceptability of large-scale biology applications such as gene drives is held in high regard by the international research community. The development of gene drives to suppress invasive and pest species and improvements to the sustainability of food systems are examples of integrative biology applications in engineering and ecology with the potential for large-scale research impact. Despite a global collective intention to ensure disruptive technologies are in broad alignment with wider social and public values, evidence of applied research organizations integrating the knowledge acquired from social research is hard to find. Concrete mechanisms to ensure public perspectives affect science decision-making are yet to emerge. We offer avenues for making inroads in what we identify as a remaining gap in public engagement research in the fields of synthetic biology and bioengineering.

The Use of Cell-free Protein Synthesis to Push the Boundaries of Synthetic Biology.

Cell-free protein synthesis is emerging as a powerful tool to accelerate the progress of synthetic biology. Notably, cell-free systems that harness extracted synthetic machinery of cells can address many of the issues associated with the complexity and variability of living systems. In particular, cell-free systems can be programmed with various configurations of genetic information, providing great flexibility and accessibility to the field of synthetic biology. Empowered by recent progress, cell-free systems are now evolving into artificial biological systems that can be tailored for various applications, including on-demand biomanufacturing, diagnostics, and new materials design. Here, we review the key developments related to cell-free protein synthesis systems, and discuss the future directions of these promising technologies.

Bidirectional hybrid erythritol-inducible promoter for synthetic biology in Yarrowia lipolytica

BackgroundThe oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica.ResultsThis study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter’s strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use.ConclusionsIn this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.

Synthetic Biology Design as a Paradigm Shift toward Manufacturing Affordable Adeno-Associated Virus Gene Therapies.

Gene therapy has demonstrated enormous potential for changing how we combat disease. By directly engineering the genetic composition of cells, it provides a broad range of options for improving human health. Adeno-associated viruses (AAVs) represent a leading gene therapy vector and are expected to address a wide range of conditions in the coming decade. Three AAV therapies have already been approved by the FDA to treat Leber's congenital amaurosis, spinal muscular atrophy, and hemophilia B. Yet these therapies cost around $850,000, $2,100,000, and $3,500,000, respectively. Such prices limit the broad applicability of AAV gene therapy and make it inaccessible to most patients. Much of this problem arises from the high manufacturing costs of AAVs. At the same time, the field of synthetic biology has grown rapidly and has displayed a special aptitude for addressing biomanufacturing problems. Here, we discuss emerging efforts to apply synthetic biology design to decrease the price of AAV production, and we propose that such efforts could play a major role in making gene therapy much more widely accessible.

Troubleshooting: The Automation of Synthetic Biology and the Labor of Technological Futures

Science and Technology Studies has long characterized the dynamics of imagining new technology as the primary driver of progress. Among these dynamics are the performative effects of the future, which have been characterized largely at the level of broader political economies. However, performative futures also operate at more intimate scales, like organizational work practices. So much is the case in the field of synthetic biology, which aims to advance biological research by making laboratory work more efficient with new digital and robotic infrastructure. This paper shows how these infrastructures have transformed laboratory work, drawing on interviews and observations with laboratory technicians in three synthetic biology facilities. These infrastructures have not eliminated laboratory work so much as introduced new kinds of skilled work. Much of this skilled work falls into the category of “troubleshooting:” the improvised correction of unexpected problems in a technical system. Organizational management treats the necessity of troubleshooting as a temporary stage in the development of its technology, while routinizing troubleshooting as a part of daily operations. This tension highlights how labor can be obscured not just by the materiality of infrastructures but by temporality—the possibility of skilled work’s obsolescence in an eventual future.

Protein Cages and Nanostructures Constructed from Protein Nanobuilding Blocks.

Protein cages and nanostructures are promising biocompatible medical materials, such as vaccines and drug carriers. Recent advances in designed protein nanocages and nanostructures have opened up cutting-edge applications in the fields of synthetic biology and biopharmaceuticals. A simple approach for constructing self-assembling protein nanocages and nanostructures is the design of a fusion protein composed of two different proteins forming symmetric oligomers. In this chapter, we describe the design and methods of protein nanobuilding blocks (PN-Blocks) using a dimeric de novo protein WA20 to construct self-assembling protein cages and nanostructures. A protein nanobuilding block (PN-Block), WA20-foldon, was developed by fusing an intermolecularly folded dimeric de novo protein WA20 and a trimeric foldon domain from bacteriophage T4 fibritin. The WA20-foldon self-assembled into several oligomeric nanoarchitectures in multiples of 6-mer. De novo extender protein nanobuilding blocks (ePN-Blocks) were also developed by fusing tandemly two WA20 with various linkers, to construct self-assembling cyclized and extended chain-like nanostructures. These PN-Blocks would be useful for the construction of self-assembling protein cages and nanostructures and their potential applications in the future.

PCR-Based Assembly of Gene Sequences by Thermodynamically Balanced Inside-Out (TBIO) Gene Synthesis.

The ability to enzymatically assemble DNA oligonucleotides into longer DNA duplexes in a process known as gene synthesis has wide-ranging applications in the fields of genetic engineering and synthetic biology. Thermodynamically balanced inside-out (TBIO) gene synthesis is one of several PCR-based primer extension genesynthesis protocols that have been developed. In TBIO gene synthesis, overlapping primers with equivalent melting temperatures (Tms) are designed so that the 5' half of the DNA is encoded by sense primers and the 3' half of the DNA molecule is encoded by antisense primers. Primer extension is initiated at the center of the DNA and continues bidirectionally to progressively elongate the DNA molecule. Here we provide the protocols necessary for performing TBIO gene synthesis to generate a DNA molecule of interest.

Opportunities for engineering outer membrane vesicles using synthetic biology approaches

Gram-negative bacteria naturally shed lipid vesicles, which contain complex molecular cargoes, from their outer membrane. These outer membrane vesicles (OMVs) have important biological functions relating to microbial stress responses, microbiome regulation, and host-pathogen interactions. OMVs are also attractive vehicles for delivering drugs, vaccines, and other therapeutic agents because of their ability to interact with host cells and their natural immunogenic properties. OMVs are also set to have a positive impact on other biotechnological and medical applications including diagnostics, bioremediation, and metabolic engineering. We envision that the field of synthetic biology offers a compelling opportunity to further expand and accelerate the foundational research and downstream applications of OMVs in a range of applications including the provision of OMV-based healthcare technologies. In our opinion, we discuss how current and potential future synergies between OMV research and synthetic biology approaches might help to further accelerate OMV research and real-world applications for the benefit of animal and human health.

Harnessing synthetic biology to engineer organoids and tissues.

The development of an organism depends on intrinsic genetic programs of progenitor cells and their spatiotemporally complex extrinsic environment. Exvivo generation of organoids from progenitor cells provides a platform for recapitulating and exploring development. Current approaches rely largely on soluble morphogens or engineered biomaterials to manipulate the physical environment, but the emerging field of synthetic biology provides a powerful toolbox to genetically manipulate cell communication, adhesion, and even cell fate. Applying these modular tools to organoids should lead to a deeper understanding of developmental principles, improved organoid models, and an enhanced capability to design tissues for regenerative purposes.

Solid-Phase Cell-Free Protein Synthesis and Its Applications in Biotechnology.

Cell-free systems for the in vitro production of proteins have revolutionized the synthetic biology field. In the last decade, this technology is gaining momentum in molecular biology, biotechnology, biomedicine and even education. Materials science has burst into the field of in vitro protein synthesis to empower the value of existing tools and expand its applications. In this sense, the combination of solid materials (normally functionalized with different biomacromolecules) together with cell-free components has made this technology more versatile and robust. In this chapter, we discuss the combination of solid materials with DNA and transcription-translation machinery to synthesize proteins within compartments, to immobilize and purify in situ the nascent protein, to transcribe and transduce DNAs immobilized on solid surfaces, and the combination of all or some of these strategies.

Periodic spatial patterning with a single morphogen.

During multicellular development, periodic spatial patterning systems generate repetitive structures, such as digits, vertebrae, and teeth. Turing patterning provides a foundational paradigm for understanding such systems. The simplest Turing systems are believed to require at least two morphogens to generate periodic patterns. Here, using mathematical modeling, we show that a simpler circuit, including only a single diffusible morphogen, is sufficient to generate long-range, spatially periodic patterns that propagate outward from transient initiating perturbations and remain stable after the perturbation is removed. Furthermore, an additional bistable intracellular feedback or operation on a growing cell lattice can make patterning robust to noise. Together, these results show that a single morphogen can be sufficient for robust spatial pattern formation and should provide a foundation for engineering pattern formation in the emerging field of synthetic developmental biology.