Piecing together a puzzle

Paving the way for synthetic biology‐based bioremediation in Europe

The emerging field of synthetic biology seems to have a great potential to the development of new biotechnologies and the understanding of self-organizing principle in life. Currently, several synthetic biologists aim to understand the evolution of naturally occurring biological systems through a bottom-up approach in contrast to the conventional reductive approach. Several lines of pioneering works have been demonstrated in that behaviors of programmed synthetic networks are predicted in vitro. However, it is crucial to create new molecular parts for (re)constructing synthetic genetic networks in artificial cell-like compartments, such as giant liposomes. We focus on RNA and RNP (ribonucleoprotein) that hold promise as new parts for synthetic biology. They are constructed with molecular design and an experimental evolution technique. So far, designed self-folding RNAs, RNA (RNP) enzymes, and nanoscale RNA architectures have been successfully constructed by utilizing Watson-Crick base-pairs together with specific RNA-RNA or RNA-protein binding motifs of known defined 3D structures. In addition, we also demonstrated that the cell-sized liposomes can be prepared by using phospholipid-coated micro-droplets, which were generated by emulsification or microfluidic techniques. We encapsulated cell-free translation system (PURE SYSTEM) in newly developed liposomes, and successfully monitored protein expression kinetics within individual liposomes in real time. By combining RNA/protein in vitro evolution techniques with giant liposome-based systems, it may be possible to generate artificial protocells with translational regulatory systems.

Synthetic biology opens up the possibility of producing new entities not found in nature, whose classification as organisms or machines has been debated. In this paper we are focusing on the delimitation of the moral value of synthetic products, in order to establish the ethically right way to behave towards them. In order to do so, we use personalism as our ethical framework. First, we examine how we can distinguish between organisms and machines. Next, we discuss whether the products of synthetic biology can be considered organisms at all and assess what their moral value is and how should we behave towards them. Finally, we discuss the hypothetical case of synthetic humans.

With the dwindling oil prices around the world and the realization that the world’s oil supply is limited, the quest for alternative fuels began and researchers along with policy makers initiated economical ways to produce ethanol, preferably from bioresources. The integration of agro-energy crops and advance technologies offers the potential for the development of cost-effective substrate for sustainable biofuel production. To develop renewable and sustainable biofuel from lignocellulosic polysaccharides or energy grasses, several pathway engineering approaches have been reported. These include the generation of new metabolic pathways by combining genes from distinct organisms in desired host to synthesize products. In the post-genomic era, synthetic biology seeks to model and construct biological components, functions, and organisms that do not exist in nature or to redesign existing biological systems to perform new functions. Even model plants are being investigated and modified to improve the enzymatic deconstruction potential of lignocellulosics into biofuel. Therefore, metabolic engineering approaches in synthetic biology are converging with synthetic genomics. Here we intend to discuss technological advancement and its probable role in the development of economically feasible biofuel. The techniques include protein engineering, imaging, fermentation technology, chromatography, enzyme technology, chemical engineering, genome sequencing, bioinformatics, and synthetic biology.

RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.

Sense and sensibility: of synthetic biology and the redesign of bioreporter circuits.

The idea of compartmentalization of genotype and phenotype in cells is key for enabling Darwinian evolution. This contribution describes bioinspired systems that use in vitro compartments—water-in-oil droplets and gel-shell beads—for the directed evolution of functional proteins. Technologies based on these principles promise to provide easier access to protein-based therapeutics, reagents for processes involving enzyme catalysis, parts for synthetic biology and materials with biological components.

Synthetic biology and metabolic engineering strategies in identifying and producing plant natural products; with emphasis on the CRISPR/Cas systems

In recent years classic genetic and molecular biology strategies (Bioballistics, Agrobacterium tumefaciens transformation, recombinant enzymes) for production of natural compounds or even breeding of medicinal and aromatic plants have expanded and improved productivity of plant-derived fine chemicals. Among those high value natural products with medicinal and cosmetic purpose (e. g., essential oils, paclitaxel, artemisinine, Vinca-Alkaloids) play a major role. Applying genetic and biotechnological techniques like metabolic engineering, site directed mutagenesis, and pathway optimization for plant optimization to reduce costs and increase productivity are in the main focus of academia and industry. Because of some drawbacks with plant cell cultures and isolated enzymes giving no sufficient high production for commercialization, research strategies shifted more and more to metabolic engineering. From the past, engineering a microorganism is proven as a valuable tool and concepts have been transferred to plant science and opened new promising perspectives for improving plants and cell lines. First, engineering crop plants was conducted, but applying these techniques for medicinal plants is rather new and has not yet been explored so well. Today cloning and expression of multiple genes in polycistronic vectors and genomic integration is of high interest and allows the reconstitution of biosynthetic pathways in heterologous organisms either plants or microorganisms. Combining science and engineering in this research field was claimed as Combinatorial Biosynthesis and later as Biology. Synthetic biology includes a large number of subareas, including enzymology, protein assembly and interactions, metabolomics, gene regulation, signal transduction and computational biology and is considered as a future approach for biotechnological plant optimization. The possibilities show exciting perspectives for the exploitation of medicinal and aromatic plants to increase the level of wanted natural products, gain insight in metabolic pathways even for new biosimilar chemicals, to improve nutrional and health promoting effects of food (nutraceuticals), and to reduce the amount of unwanted by-products with potential toxic or allergic activities.

BackgroundSynthetic biological systems are currently created by an ad-hoc, iterativeprocess of specification, design, and assembly. These systems would greatlybenefit from a more formalized and rigorous specification of the desiredsystem components as well as constraints on their composition. Therefore,the creation of robust and efficient design flows and tools is imperative.We present a human readable language (Eugene) that allows for thespecification of synthetic biological designs based on biological parts, aswell as provides a very expressive constraint system to drive the automaticcreation of composite Parts (Devices) from a collection of individualParts.ResultsWe illustrate Eugene's capabilities in three different areas: Devicespecification, design space exploration, and assembly and simulationintegration. These results highlight Eugene's ability to createcombinatorial design spaces and prune these spaces for simulation orphysical assembly. Eugene creates functional designs quickly andcost-effectively.ConclusionsEugene is intended for forward engineering of DNA-based devices, and throughits data types and execution semantics, reflects the desired abstractionhierarchy in synthetic biology. Eugene provides a powerful constraint systemwhich can be used to drive the creation of new devices at runtime. Itaccomplishes all of this while being part of a larger tool chain whichincludes support for design, simulation, and physical device assembly.

Chapter 1 - Systems biology, synthetic biology, and metabolic engineering

The last two decades have seen vigorous activity in synthetic biology research and the ever-increasing applications of these technologies. However, pedagogical research pertaining to teaching synthetic biology is scarce, especially when compared to other science and engineering disciplines. Within Canada, there are only three universities that offer synthetic biology programs, two of which are at the undergraduate level. Rather than taking place in formal academic settings, many Canadian undergraduate students are introduced to synthetic biology through participation in the annual International Genetically Engineered Machine (iGEM) competition. Although the iGEM competition has had a transformative impact on synthetic biology training in other nations, its impact in Canada has been relatively modest. Consequently, the iGEM competition remains a major setting for synthetic biology education in Canada. To promote further development of synthetic biology education, we surveyed undergraduate students from the Canadian iGEM design teams of 2019. We extracted insights from these data using qualitative analysis to provide recommendations for best teaching practices in synthetic biology undergraduate education, which we describe through our proposed Framework for Transdisciplinary Synthetic Biology Education (FTSBE).

In May 2010, researchers at the J. Craig Venter Institute announced the creation of the world’s fi rst synthetic organism—a bacterial host cell whose self-replicating genome was human-made. This momentous achievement raises questions regarding the potential risks and benefi ts of synthesizing genomes, and eventually, organisms. According to proponents, synthetic biology offers great promise. Some scientists suggest that the emerging fi eld could lead to advancements in individualized medicine, more effi cient vaccine and drug production, new renewable energy sources, higher-yielding and more sustainable crops, and organisms that can remediate harmful chemicals in the environment. Synthetic biology is also widely acknowledged to have the potential to adversely affect human health, the environment, and national security. The possibility for unintended environmental effects concerns Allison Snow, a professor in the Department of Evolution, Ecology, and Organismal Biology at the Ohio State University. Uncontrolled escape of synthetic organisms and the rise of new invasive species are a few of the threats, according to Snow. Given the risks, synthetic organisms will need to be thoroughly evaluated before they are moved outside contained facilities. “Ecological research takes time and funding,” Snow said during a public presentation on synthetic biology in July 2010. “This is why risk assessment research shouldn’t be left for the last minute. It should go in tandem as the development of these products is moving forward.” Policymakers recognize there are trade-offs between scientifi c advancement and societal hazard. The same day the Venter Institute’s research was published, President Barack Obama directed his newly minted Presidential Commission for the Study of Bioethical with just a few new traits—are widely considered to pose few new risks. With synthetic biology, which is an extension of genetic engineering, new GMOs like blue-green algae for biofuels could be more challenging to evaluate. We have less experience with these organisms and the newly invented traits they will have. It would be naive to assume that regulations will catch every bad idea and prevent it from happening.” Experts in ethics, biosecurity, and law also see cause for concern. Some have raised doubts about society’s ability to plan for low-probability, highimpact events, such as the release of a synthetic organism. Doubts linger about gaps in the current regulatory framework to oversee privately funded research, especially that of “do-it-yourselfers” who work outside of agencies, universities, and corporations. Even if the laws were suffi cient to cover synthetic biology products, the more important issue, warns Michael Rodemeyer, is “whether the agencies have the resources and tools they need to both assess the risks of this new technology and to manage the risks as well.” Now at the University of Virginia, Rodemeyer was formerly assistant director for environment at the White House Offi ce of Science and Technology Policy. “The challenge, though, particularly as the technology develops will be to develop guidelines that are suffi ciently cautionary, but without imposing unnecessarily expensive and cumbersome containment requirements that might hinder research.” It remains to be seen whether or how the federal government will strike a balance between scientifi c discovery and risk mitigation.

Synthetic genomics is a newly emerging area of synthetic biology that allows us to design, synthesize, and construct a desired genome to create new synthetic life forms. It has been possible because of recently developed advanced cloning and DNA synthesis technologies. Synthetic genomes of bacteria, viruses, and yeast have been chemically synthesized and characterized. Synthetic viruses have been designed and constructed in a way that alters the pathogenicity but maintains antigenicity for vaccination purpose. Mycoplasma mycoides JCVI-syn1.0, JCVI-syn2.0, and JCVI-syn3.0 and yeast Saccharomyces cerevisiae chromosome have been chemically synthesized and grown as fully functional cells. Synthetic genome technology allows the installation of complete biosynthetic pathways, deletion of non-essential genes, and replacement of the defective chromosome with synthetic chromosome for proper cell functioning. The chapter highlights the recent progress, challenges and opportunities for accelerating synthetic genomics toward biomedical, therapeutic, and industrial applications.

In recent years, the publication of the studies on the transmissibility in mammals of the H5N1 influenza virus and synthetic genomes has triggered heated and concerned debate within the community of scientists on biological dual-use research; these papers have raised the awareness that, in some cases, fundamental research could be directed to harmful experiments, with the purpose of developing a weapon that could be used by a bioterrorist. Here is presented an overview regarding the dual-use concept and its related international agreements which underlines the work of the Australia Group (AG) Export Control Regime. It is hoped that the principles and activities of the AG, that focuses on export control of chemical and biological dual-use materials, will spread and become well known to academic researchers in different countries, as they exchange biological materials (i.e. plasmids, strains, antibodies, nucleic acids) and scientific papers. To this extent, and with the aim of drawing the attention of the scientific community that works with yeast to the so called Dual-Use Research of Concern, this article reports case studies on biological dual-use research and discusses a synthetic biology applied to the yeast Saccharomyces cerevisiae, namely the construction of the first eukaryotic synthetic chromosome of yeast and the use of yeast cells as a factory to produce opiates. Since this organism is considered harmless and is not included in any list of biological agents, yeast researchers should take simple actions in the future to avoid the sharing of strains and advanced technology with suspicious individuals.