Field Of Synthetic Biology Research Articles

A Concise Review on Toxicity and Pharmacological Aspects of Foeniculum vulgare with Emphasis on Anti-Cancer Potential

The recent advancements in field of genomics and synthetic biology has paved the way for determination and utilization of pharmaceutical properties of beneficial plants. Foeniculum vulgare is a member of Apiaceae family, commonly known as fennel. It is globally distributed aromatic medicinal herb. F. vulgare falls in the category of World’s most important medicinal herb because of its economic status and pharmaceutical industrial applications. It has been used as herbal treatment for a longer period of time as an effective medicine to cure different ailments such as liver pain, kidney disorders, swollen stomach, abdominal pain, and mouth ulcerand no documented severe side effects reported. Essential oil, flavonoids and phenolic compounds are major chemical constituents of F.vulgare. Volatile and non-volatile compounds of this plant are responsible for its biological activities. The Pharmacological experiments performed by in vitro and in vivo models, revealed that F. vulgare has strong therapeutic effects. This review is supposed to shed the light on anti-bacterial, antioxidant, antidiabetic, antifungal, antimicrobial, antianxiety, hepatoprotective, memory protective, acaricidal, anti-cancer, anti-inflammatory and anti-mutagenic activities.

Rapid HILIC-Z ion mobility mass spectrometry (RHIMMS) method for untargeted metabolomics of complex biological samples

IntroductionRecent advances in high-throughput methodologies in the ‘omics’ and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of complex biological samples.ObjectiveThe objective of this research was to evaluate a straightforward to implement LC–MS metabolomics method using a commercially available chromatography column that provides increased throughput. Reducing run time can potentially impact chromatography and therefore the effects of ion mobility spectrometry to expand peak capacity were also evaluated. Additional confidence provided via collision cross section measurements for detected features was also explored.MethodsA rapid untargeted metabolomics workflow was developed with broad metabolome coverage, combining zwitterionic-phase hydrophilic interaction chromatography (HILIC-Z) with drift tube ion mobility-quadrupole time-of-flight (DTIM-qTOF) mass spectrometry. The analytical performance of our method was explored using extracts from complex biological samples, including a reproducibility study on chicken serum and a simple comparative study on a bacterial metabolome.ResultsThe method is acronymised RHIMMS for rapid HILIC-Z ion mobility mass spectrometry. We present the RHIMMS workflow starting with data acquisition, followed by data processing and analysis. RHIMMS demonstrates improved chromatographic separation for a selection of metabolites with wide physicochemical properties while maintaining reproducibility at better than 20% over 200 injections at 3.5 min per sample for the selected metabolites, and a mean of 13.9% for the top 50 metabolites by intensity. Additionally, the combination of rapid chromatographic separation with ion mobility allows improved annotation and the ability to distinguish isobaric compounds.ConclusionOur results demonstrate RHIMMS to be a rapid, reproducible, sensitive and high-resolution analytical platform that is highly applicable to the untargeted metabolomics analysis of complex samples.

From Sharks to Yeasts: Squalene in the Development of Vaccine Adjuvants.

Squalene is a natural linear triterpene that can be found in high amounts in certain fish liver oils, especially from deep-sea sharks, and to a lesser extent in a wide variety of vegeTable oils. It is currently used for numerous vaccine and drug delivery emulsions due to its stability-enhancing properties and biocompatibility. Squalene-based vaccine adjuvants, such as MF59 (Novartis), AS03 (GlaxoSmithKline Biologicals), or AF03 (Sanofi) are included in seasonal vaccines against influenza viruses and are presently being considered for inclusion in several vaccines against SARS-CoV-2 and future pandemic threats. However, harvesting sharks for this purpose raises serious ecological concerns that the exceptional demand of the pandemic has exacerbated. In this line, the use of plants to obtain phytosqualene has been seen as a more sustainable alternative, yet the lower yields and the need for huge investments in infrastructures and equipment makes this solution economically ineffective. More recently, the enormous advances in the field of synthetic biology provided innovative approaches to make squalene production more sustainable, flexible, and cheaper by using genetically modified microbes to produce pharmaceutical-grade squalene. Here, we review the biological mechanisms by which squalene-based vaccine adjuvants boost the immune response, and further compare the existing sources of squalene and their environmental impact. We propose that genetically engineered microbes are a sustainable alternative to produce squalene at industrial scale, which are likely to become the sole source of pharmaceutical-grade squalene in the foreseeable future.

Shifting Perspectives: A cinematic dialogue about Synthetic Biology in a more-than-human world

The short experimental film Shifting Perspectives stems from a collaborative research project initiated in 2019 in Sydney, Australia, during the ‘Choreographic Hack Lab'—a week-long laboratory co-presented by Critical Path and Sydney Festival in partnership with the Museum of Applied Arts and Sciences (MAAS), which asked artists and academics to rethink and respond to the idea of the Anthropocene (Pini & George, 2019). The film was later developed in 2020 during a Responsive Residency at Critical Path, Sydney, awarded to anthropologist and choreographer Sarah Pini in collaboration with Jestin George, biotechnologist and visual artist, and Melissa Ramos, visual artist and filmmaker. This work aims to open a multivocal interdisciplinary dialogue across screendance, performance and synthetic biology. Inspired by a recent conversation between two leaders in the field of synthetic biology (Sarah Richardson and Tom Knight) and their divergent approaches to working with microbial life (Agapakis, 2019), the film invites to consider how collaborating with microorganisms can reshape our future in a more-than-human world. 

Sequential assembly and separation of synthetic cell models using microfluidics

The emergent field of bottom-up synthetic biology aims to recombine diverse biological systems in artificial cell models for studying complex cellular phenomena in isolation. With dimensions and membrane that resemble biological cells, giant unilamellar vesicles (GUVs) are established as a promising biomimetic compartment for constructing synthetic cells from scratch. However, one of the main reasons that restricts the broad use of GUV-based cell models for studying complex biomolecular systems in vitro, emanates from the inevitable generation of residual components (e.g., surfactants, oil droplets, polymers, nanometric lipid vesicles, etc.) during the formation and manipulation of GUVs.

Bioengineering for the industrial production of 2,3-butanediol by the yeast, Saccharomyces cerevisiae.

Owing to issues, such as the depletion of petroleum resources and price instability, the development of biorefinery related technologies that produce fuels, electric power, chemical substances, among others, from renewable resources is being actively promoted. 2,3-Butanediol (2,3-BDO) is a key compound that can be used to produce various chemical substances. In recent years, 2,3-BDO production using biological processes has attracted extensive attention for achieving a sustainable society through the production of useful compounds from renewable resources. With the development of genetic engineering, metabolic engineering, synthetic biology, and other research field, studies on 2,3-BDO production by the yeast, Saccharomyces cerevisiae, which is safe and can be fabricated using an established industrial-scale cultivation technology, have been actively conducted. In this review, we sought to describe 2,3-BDO and its derivatives; discuss 2,3-BDO production by microorganisms, in particular S. cerevisiae, whose research and development has made remarkable progress; describe a method for separating and recovering 2,3-BDO from a microbial culture medium; and propose future prospects for the industrial production of 2,3-BDO by microorganisms.

Construction of Strong Promoters by Assembling Sigma Factor Binding Motifs.

Development of strong promoters is of growing interest in the field of biotechnology and synthetic biology. Here we present a protocol for the construction of strong prokaryotic promoters that can be recognized by designated multiple sigma factors by interlocking their cognate binding motifs on DNA strands. Strong and stress responsive promoters for Escherichia coli and Bacillus subtilis have been created following the presented protocol. Customized promoters could be easily developed for fine-tuning gene expression or overproducing enzymes with prokaryotic cell factories.

Implementing Multi-Enzyme Biocatalytic Systems Using Nanoparticle Scaffolds.

Interest in multi-enzyme synthesis outside of cells (in vitro) is becoming far more prevalent as the field of cell-free synthetic biology grows exponentially. Such synthesis would allow for complex chemical transformations based on the exquisite specificity of enzymes in a "greener" manner as compared to organic chemical transformations. Here, we describe how nanoparticles, and in this specific case-semiconductor quantum dots, can be used to both stabilize enzymes and further allow them to self-assemble into nanocomplexes that facilitate high-efficiency channeling phenomena. Pertinent protocol information is provided on enzyme expression, choice of nanoparticulate material, confirmation of enzyme attachment to nanoparticles, assay format and tracking, data analysis, and optimization of assay formats to draw the best analytical information from the underlying processes.

Establishing a Cell-Free Transcription-Translation Platform for Cutibacterium acnes to Prototype Engineered Metabolic and Synthetic Biology.

In the past few years, new bacterial-cell-free transcription-translation systems have emerged as potent and quick platforms for protein production as well as for prototyping of DNA regulatory elements, genetic circuits, and metabolic pathways. The Gram-positive commensal Cutibacterium acnes is one of the most abundant bacteria present in the human skin microbiome. However, it has recently been reported that some C. acnes phylotypes can be associated with common inflammatory skin conditions, such as acne vulgaris, whereas others seem to play a protective role, acting as possible "skin probiotics". This fact has made C. acnes become a bacterial model of interest for the cosmetic industry. In the present study we report for the first time the development and optimization of a C. acnes-based cell-free system (CFS) that is able to produce 85 μg/mL firefly luciferase. We highlight the importance of harvesting the bacterial pellet in mid log phase and maintaining CFS reactions at 30 °C and physiological pH to obtain the optimal yield. Additionally, a C. acnes promoter library was engineered to compare coupled in vitro TX-TL activities, and a temperature biosensor was tested, demonstrating the wide range of applications of this toolkit in the synthetic biology field.

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8.

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

Bioinspired Networks of Communicating Synthetic Protocells.

The bottom-up synthesis of cell-like entities or protocells from inanimate molecules and materials is one of the grand challenges of our time. In the past decade, researchers in the emerging field of bottom-up synthetic biology have developed different protocell models and engineered them to mimic one or more abilities of biological cells, such as information transcription and translation, adhesion, and enzyme-mediated metabolism. Whilst thus far efforts have focused on increasing the biochemical complexity of individual protocells, an emerging challenge in bottom-up synthetic biology is the development of networks of communicating synthetic protocells. The possibility of engineering multi-protocellular systems capable of sending and receiving chemical signals to trigger individual or collective programmed cell-like behaviours or for communicating with living cells and tissues would lead to major scientific breakthroughs with important applications in biotechnology, tissue engineering and regenerative medicine. This mini-review will discuss this new, emerging area of bottom-up synthetic biology and will introduce three types of bioinspired networks of communicating synthetic protocells that have recently emerged.

Accelerating Whole-Cell Simulations of mRNA Translation Using a Dedicated Hardware

In recent years, intracellular biophysical simulations have been used with increasing frequency not only for answering basic scientific questions but also in the field of synthetic biology. However, since these models include networks of interaction between millions of components, they are extremely time-consuming and cannot run easily on parallel computers. In this study, we demonstrate for the first time a novel approach addressing this challenge by using a dedicated hardware designed specifically to simulate such processes. As a proof of concept, we specifically focus on mRNA translation, which is the process consuming most of the energy in the cell. We design a hardware that simulates translation in Escherichia coli and Saccharomyces cerevisiae for thousands of mRNAs and ribosomes, which is in orders of magnitude faster than a similar software solution. With the sharp increase in the amount of genomic data available today and the complexity of the corresponding models inferred from them, we believe that the strategy suggested here will become common and can be used among others for simulating entire cells with all gene expression steps.

Abstract PO-033: Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) with systemic chemotherapeutic drugs has remained a challenge, due in part to the hypovascularized and poorly perfused nature of PDAC tumors. Limited blood flow within the tumor tissue creates an extremely hypoxic microenvironment and impedes the accumulation of drugs. Moreover, local immunosuppression in PDAC has so far limited the efficacy of immunotherapy approaches. However, these very features that have interfered with systemic therapy in PDAC (hypoperfusion, hypoxia, and immunosuppression) are potential advantages for the use of bacterial therapies. Bacteria have been used as a directed cancer therapy for over 100 years, starting with Dr. William Coley’s use of heat killed bacteria (Coley’s Toxin) against sarcomas in 1893. Recent developments in the field of synthetic biology have made it possible to engineer complex logical circuits into bacteria, enabling the manufacture of anticancer therapies directly within the tumor parenchyma. Bacteria can actively migrate through tissues, they can thrive in hypoxic microenvironments, and they benefit from the local immune suppression. We have therefore worked to develop novel bacterial strains for targeting PDAC. We began by testing a range of bacteria-derived toxins that could be used as a payload to target PDAC. These toxins were produced by an engineered strain of a non-toxic, probiotic E. coli Nissle 1917. We identified four pore-forming toxins that significantly reduced viability of the cells compared to bacterially produced GFP: hemolysin E, heat stable enterotoxin, magainin, and theta toxin. We then performed a secondary screen using a novel PDAC explant model system developed in our lab, in which thick slices of murine or human PDAC are culture intact for up to 7 days. Consistent with the monolayer screen, two of the candidate compounds, heat stable enterotoxin and theta toxin, significantly increased tissue death compared to non-toxic GFP-producing bacteria. Finally, we delivered live bacteria producing either toxins or GFP into KPC mouse tumors through intratumoral injection. While GFP-producing strains did not induce a change in tumor growth kinetics, theta toxin treatment demonstrated an immediate, prolonged stabilization of tumor volume for more than a month. Histological analyses of treated tumors demonstrated that diffuse populations of bacteria co-localized with regions of tumor necrosis and cell death. Most interestingly, while there was minimal spread of bacteria to healthy non-target tissues, translocation of the bacteria did occur to regions of liver metastases and secondary papilloma tumors, suggesting a mechanism for diffuse treatment of known and unknown metastases following the initial treatment of the primary tumor. Together these studies demonstrate that cytotoxic bacterial therapy is an effective candidate strategy to circumvent the difficulties in systemic treatment of PDAC. Citation Format: Amanda R. Decker, Tetsuhiro Harimoto, Steve A. Sastra, Tal Danino, Kenneth P. Olive. Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-033.

A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models.

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residues—which range in size and chemical composition—with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

Xanthopsin-Like Systems via Site-Specific Click-Functionalization of a Retinoic Acid Binding Protein.

The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the in vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.

Plastics biodegradation and recycling - the introduction of China-Europe cooperation project "Synthetic microorganism communities for plastic degradation and transformation"

The China-European environmental biotechnology cooperation research project on the biodegradation of waste plastics is jointly funded by the National Natural Science Foundation of China (NSFC) and the European Commission (EC), and aims to encourage Chinese and European scientists to carry out substantive research in the field of "Microorganism communities for plastics biodegradation". The goal of the project is to use the metabolic capacity of microbial communities to degrade petrochemical plastics that are easy to cause environmental pollution into monomers and small molecules, thereby realizing the biosynthesis of high-value biochemicals by microorganisms. This can not only solve the problem of plastic pollution, but also "turn waste into treasure" and create higher economic benefits. The China-European cooperative research project will promote in-depth cooperation between scientists from both sides in the field of synthetic biology, and help the two sides establish long-term and stable international exchanges and cooperation. Both China and the EU will work to solve the global plastic pollution problem, form a strategic force of science and technology, and jointly open a new chapter in the field of resource utilization of non-degradable plastics.

Engineering living therapeutics with synthetic biology.

The steadfast advance of the synthetic biology field has enabled scientists to use genetically engineered cells, instead of small molecules or biologics, as the basis for the development of novel therapeutics. Cells endowed with synthetic gene circuits can control the localization, timing and dosage of therapeutic activities in response to specific disease biomarkers and thus represent a powerful new weapon in the fight against disease. Here, we conceptualize how synthetic biology approaches can be applied to programme living cells with therapeutic functions and discuss the advantages that they offer over conventional therapies in terms of flexibility, specificity and predictability, as well as challenges for their development. We present notable advances in the creation of engineered cells that harbour synthetic gene circuits capable of biological sensing and computation of signals derived from intracellular or extracellular biomarkers. We categorize and describe these developments based on the cell scaffold (human or microbial) and the site at which the engineered cell exerts its therapeutic function within its human host. The design of cell-based therapeutics with synthetic biology is a rapidly growing strategy in medicine that holds great promise for the development of effective treatments for a wide variety of human diseases.

Directed Signaling Cascades in Monodisperse Artificial Eukaryotic Cells.

The bottom-up assembly of multicompartment artificial cells that are able to direct biochemical reactions along a specific spatial pathway remains a considerable engineering challenge. In this work, we address this with a microfluidic platform that is able to produce monodisperse multivesicular vesicles (MVVs) to serve as synthetic eukaryotic cells. Using a two-inlet polydimethylsiloxane channel design to co-encapsulate different populations of liposomes we are able to produce lipid-based MVVs in a high-throughput manner and with three separate inner compartments, each containing a different enzyme: α-glucosidase, glucose oxidase, and horseradish peroxidase. We demonstrate the ability of these MVVs to carry out directed chemical communication between the compartments via the reconstitution of size-selective membrane pores. Therefore, the signal transduction, which is triggered externally, follows a specific spatial pathway between the compartments. We use this platform to study the effects of enzyme cascade compartmentalization by direct analytical comparison between bulk, one-, two-, and three-compartment systems. This microfluidic strategy to construct complex hierarchical structures is not only suitable to study compartmentalization effects on biochemical reactions but is also applicable for developing advanced drug delivery systems as well as minimal cells in the field of bottom-up synthetic biology.

Exploring presentations of sustainability by US synthetic biology companies.

The field of synthetic biology is increasingly being positioned as a key driver of a more sustainable, bio-based economy, and has seen rapid industry growth over the past 15 years. In this paper we undertake an exploratory investigation of the relationship between sustainability and synthetic biology, identifying and analyzing sustainability-related language on the public websites of 24, US-based synthetic biology companies. We observe that sustainability is a visible part of the self-presentation of the nascent synthetic biology industry, explicitly mentioned by 18 of the 24 companies. The dominant framing of sustainability on these company websites emphasizes environmental gains and “free-market” approaches to sustainability, with little explicit mention of social dimensions of sustainability such as access, justice or intergenerational equity. Furthermore, the model of sustainability presented focuses on incremental transition towards environmental sustainability through direct substitution of products and processes using bioengineered alternatives (n = 16 companies), with no change in societal consumption or policy frameworks required in order to see sustainability gains. One-third of the companies analyzed (n = 8) mention “nature” on their websites, variously framing it as a resource to be managed or as a source of inspiration; whether the latter signals a potentially more complex relationship with nature than advanced free-market models of sustainability remains to be seen. As the synthetic biology industry begins to grow in size and visibility, we suggest this is an opportune time for the community to engage in explicit deliberation about its approach to sustainability.

Synergies of Systems Biology and Synthetic Biology in Human Microbiome Studies.

A number of studies have shown that the microbial communities of the human body are integral for the maintenance of human health. Advances in next-generation sequencing have enabled rapid and large-scale quantification of the composition of microbial communities in health and disease. Microorganisms mediate diverse host responses including metabolic pathways and immune responses. Using a system biology approach to further understand the underlying alterations of the microbiota in physiological and pathological states can help reveal potential novel therapeutic and diagnostic interventions within the field of synthetic biology. Tools such as biosensors, memory arrays, and engineered bacteria can rewire the microbiome environment. In this article, we review the computational tools used to study microbiome communities and the current limitations of these methods. We evaluate how genome-scale metabolic models (GEMs) can advance our understanding of the microbe–microbe and microbe–host interactions. Moreover, we present how synergies between these system biology approaches and synthetic biology can be harnessed in human microbiome studies to improve future therapeutics and diagnostics and highlight important knowledge gaps for future research in these rapidly evolving fields.