Implications Of Synthetic Biology Research Articles

Community science designed ribosomes with beneficial phenotypes

Functional design of ribosomes with mutant ribosomal RNA (rRNA) can expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts are hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we develop an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We apply our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides insights into rRNA sequence-function relationships and has implications for synthetic biology.

Evidence of a possible multicellular life cycle in Escherichia coli

Biofilms are surface-attached multicellular microbial communities. Their genetics have been extensively studied, but the cell-scale morphogenetic events of their formation are largely unknown. Here, we recorded the entirety of morphogenesis in Escherichia coli, and discovered a previously unknown multicellular self-assembly process. Unattached, single-cells formed 4-cell rosettes which grew intoconstant-width chains. After ∼10 cell generations, these multicellular chains attached to surfaces and stopped growing. Chains remained clonal throughout morphogenesis. We showed that this process generates biofilms, which we found are composed of attached clonal chains, aligned in parallel. We investigated genetics of chain morphogenesis: Ag43 facilitates rosette formation and clonality; type-1 fimbriae and curli promote stability and configuration; and extracellular polysaccharide production facilitates attachment. Our study establishes that E.coli, a unicellular organism, can follow a multistage, clonal, genetically-regulated, rosette-initiated multicellular life cycle. These findings have implications for synthetic biology, multicellular development, and the treatment and prevention of bacterial diseases.

MISCELÁNEA: BIOLOGÍA SINTÉTICA Y DERECHO INTERNACIONAL: DÉBILES CONSENSOS ANTE DESAFÍOS INMENSOS

The dizzying scientific advances in the field of biology and biotechnology, which we are collectivelly refer to as synthetic biology, have defined new frontiers of knowledge. The latest stage in this process has been the rapid mastery of genome editing techniques, mainly CRISPR/Cas9 tools and systems. This study, from the perspective of international law, deals, on the one hand, with the implications of synthetic biology on human health, thus covering the bioethics and human rights perspective in relation to gene research and gene therapy; and, on the other hand, with the responses of international law to the challenges that gene editing and the development of modified gene drives present for biodiversity. In both dimensions, the weak existing international consensus and the profound interactions in a wide range of sectors of international law, with the presence of diverse international institutional mechanisms, are apparent, which is evidence of a multiple and complex normative crossroads.

Discovery, Design, and Structural Characterization of Alkane-Producing Enzymes across the Ferritin-like Superfamily

To complement established rational and evolutionary protein design approaches, significant efforts are being made to utilize computational modeling and the diversity of naturally occurring protein sequences. Here, we combine structural biology, genomic mining, and computational modeling to identify structural features critical to aldehyde deformylating oxygenases (ADOs), an enzyme family that has significant implications in synthetic biology and chemoenzymatic synthesis. Through these efforts, we discovered latent ADO-like function across the ferritin-like superfamily in various species of Bacteria and Archaea. We created a machine learning model that uses protein structural features to discriminate ADO-like activity. Computational enzyme design tools were then utilized to introduce ADO-like activity into the small subunit of Escherichia coli class I ribonucleotide reductase. The integrated approach of genomic mining, structural biology, molecular modeling, and machine learning has the potential to be utilized for rapid discovery and modulation of functions across enzyme families.

Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels

Escherichia coli have developed one of the most efficient regulatory response mechanisms to phosphate starvation. The machinery involves a cascade with a two-component system (TCS) that relays the external signal to the genetic circuit, resulting in a feedback response. Achieving a quantitative understanding of this system has implications in synthetic biology and biotechnology, for example, in applications for wastewater treatment. To this aim, we present a computational model and experimental results with a detailed description of the TCS, consisting of PhoR and PhoB, together with the mechanisms of gene expression. The model is parameterised within the feasible range, and fitted to the dynamic response of our experimental data on PhoB as well as PhoA, the product of this network that is used in alkaline phosphatase production. Deterministic and stochastic simulations with our model predict the regulation dynamics in higher external phosphate concentrations while reproducing the experimental observations. In a cycle of simulations and experimental verification, our model predicts and explores phenotypes with various synthetic promoter designs that can optimise the inorganic phosphate intake in E. coli. Sensitivity analysis demonstrates that the Pho-controlled genes have a significant influence over the phosphate response. Together with experimental findings, our model should thus provide insights for the investigations on engineering new sensors and regulators for living technologies.

Genome Engineering for Stem Cell Transplantation.

To avoid the ethical issues of embryonic stem cells, genome engineering has focused on inducible pluripotent stem cells, which can develop into all 3 germ layers. The ability to detect methylation patterns in these cells allows research into pluripotency markers. The recently developed CRISPR system has allowed widespread application of genome engineering techniques. The CRISPR-Cas9 system, a potent system for genome editing, can be used for gene knockout or knock-in genome manipulations through substitution of a target genetic sequence with a desired donor sequence. Two types of genome engineering can be initiated: homologous or nonhomologous DNA repair by the Cas9 nuclease. Delivery of the CRISPR-Cas9 and target donor vectors in human pluripotent stem cells can be accomplished via viral and nonviral delivery methods. Nonviral delivery includes lipid-mediated transfection and electroporation. It has become the most common and efficient in vitro delivery method for human pluripotent stem cells. The CRISPR-Cas9 system can be combined with inducible pluripotent stem cells to generate single or multiple gene knockouts, correct mutations, or insert reporter transgenes. Knockouts can also be utilized to investigate epigenetic roles and targets, such as investigation of DNA methylation. CRISPR could be combined with human pluripotent stem cells to explore genetic determinants of lineage choice, differentiation, and stem cell fate, allowing investigators to study how various genes or noncoding elements contribute to specific processes and pathways. The CRISPR-Cas9 system can also be used to create null or nucleasedead Cas9, which has no enzymatic activity but has been utilized through fusion with other functional protein domains. In conclusion, RNA-guided genome targeting will have broad implications for synthetic biology, direct perturbation of gene networks, and targeted ex vivo and in vivo gene therapy.

The Security Implications of Synthetic Biology

Advances in synthetic biology hold great promise, but to minimise security threats, national and international regulation will need to keep pace.

Kernel Architecture of the Genetic Circuitry of the Arabidopsis Circadian System.

A wide range of organisms features molecular machines, circadian clocks, which generate endogenous oscillations with ~24 h periodicity and thereby synchronize biological processes to diurnal environmental fluctuations. Recently, it has become clear that plants harbor more complex gene regulatory circuits within the core circadian clocks than other organisms, inspiring a fundamental question: are all these regulatory interactions between clock genes equally crucial for the establishment and maintenance of circadian rhythms? Our mechanistic simulation for Arabidopsis thaliana demonstrates that at least half of the total regulatory interactions must be present to express the circadian molecular profiles observed in wild-type plants. A set of those essential interactions is called herein a kernel of the circadian system. The kernel structure unbiasedly reveals four interlocked negative feedback loops contributing to circadian rhythms, and three feedback loops among them drive the autonomous oscillation itself. Strikingly, the kernel structure, as well as the whole clock circuitry, is overwhelmingly composed of inhibitory, rather than activating, interactions between genes. We found that this tendency underlies plant circadian molecular profiles which often exhibit sharply-shaped, cuspidate waveforms. Through the generation of these cuspidate profiles, inhibitory interactions may facilitate the global coordination of temporally-distant clock events that are markedly peaked at very specific times of day. Our systematic approach resulting in experimentally-testable predictions provides insights into a design principle of biological clockwork, with implications for synthetic biology.

Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids.

Vulnerability at the Heart of the Ethical Implications of New Biotechnologies

Abstract Starting from research on biotechnology and its applications to living organisms, this paper presents the key features of modern-day synthetic biology, as well as its main ethical implications. The analysis of the paradox of the concept of robustness in the creation of microorganisms through synthetic biology leads us to address the topic of vulnerability, applied to man, but also to all other living beings. The concept of “enhanced human being” will strengthen the link between complexity and vulnerability as inherent features of living beings. Reflecting upon the importance of considering vulnerability applied to man’s three-fold dimensions - physical, psycho-social and spiritual - and their interaction with their environment, we will define a type of anthropology which may constitute the basis of the study on the ethical implications of synthetic biology. This will lead to present the purpose of an ethical limit to the temptation of « allmightiness », which the concept of enhanced human being could entail, and vulnerability as a defining feature of all living beings.

Extraordinarily adaptive properties of the genetically encoded amino acids.

Using novel advances in computational chemistry, we demonstrate that the set of 20 genetically encoded amino acids, used nearly universally to construct all coded terrestrial proteins, has been highly influenced by natural selection. We defined an adaptive set of amino acids as one whose members thoroughly cover relevant physico-chemical properties, or “chemistry space.” Using this metric, we compared the encoded amino acid alphabet to random sets of amino acids. These random sets were drawn from a computationally generated compound library containing 1913 alternative amino acids that lie within the molecular weight range of the encoded amino acids. Sets that cover chemistry space better than the genetically encoded alphabet are extremely rare and energetically costly. Further analysis of more adaptive sets reveals common features and anomalies, and we explore their implications for synthetic biology. We present these computations as evidence that the set of 20 amino acids found within the standard genetic code is the result of considerable natural selection. The amino acids used for constructing coded proteins may represent a largely global optimum, such that any aqueous biochemistry would use a very similar set.

Social science contributions compared in synthetic biology and nanotechnology

With growing attention to societal issues and implications of synthetic biology, we investigate sources of social science publication knowledge in synthetic biology and probe what might be learned by comparison with earlier rounds of social science research in nanotechnology. “Social science” research is broadly defined to include publications in conventional social science as well as humanities, law, ethics, business, and policy fields. We examine the knowledge clusters underpinning social science publications in nanotechnology and synthetic biology using a methodology based on the analysis of cited references. Our analysis finds that social science research in synthetic biology already has traction and direction, rooted in an ethical, legal, and social implications framework. However, compared with nanotechnology, social science research in synthetic biology could further explore opportunities and openings for engagement, anticipatory, and downstream application perspectives that will help to build a wider platform for insights into current and future societal impacts.

Signatures of Conformational Stability and Oxidation Resistance in Proteomes of Pathogenic Bacteria

Protein oxidation is known to compromise vital cellular functions. Therefore, invading pathogenic bacteria must resist damage inflicted by host defenses via reactive oxygen species. Using comparative genomics and experimental approaches, we provide multiple lines of evidence that proteins from pathogenic bacteria have acquired resistance to oxidative stress by an increased conformational stability. Representative pathogens exhibited higher survival upon HSP90 inhibition and a less-oxidation-prone proteome. A proteome signature of the 46 pathogenic bacteria encompasses 14 physicochemical features related to increasing protein conformational stability. By purifying ten representative proteins, we demonstrate invitro that proteins with a pathogen-like signature are more resistant to oxidative stress as a consequence of their increased conformational stability. A compositional signature of the pathogens' proteomes allowed the design of protein fragments more resilient to both unfolding and carbonylation, validating the relationship between conformational stability and oxidability with implications for synthetic biology and antimicrobial strategies.

The Living Regulatory Challenges of Synthetic Biology

IntroductionSynthetic biology is one of the fastest developing and most promising emerging technologies.1 It will permit scientists to design living organisms unlike any found in nature and to redesign existing organisms to have enhanced or novel qualities.2 3 4 5 While traditional biotechnology involves the transfer of a small amount of genetic material from one species to another, synthetic biology will permit the purposeful assembly of an entire organism. Synthetically designed organisms, it is hoped, might be put to myriad beneficial uses, including better detection and treatment of disease, the remediation of environmental pollutants, and the production of new sources of energy, medicines, and other valuable products.3 Engineered life forms, however, also might pose risks to human health and the environment. Exactly what those hazards are and how they might be controlled cannot be fully determined in advance of the very research necessary to develop this novel science in the first instance.This Article discusses potential regulatory challenges under the existing U.S. regulatory system concerning the first synthetic biology organisms that are anticipated to be commercialized. Much of the policy and ethical commentary on synthetic biology to date has focused on biosecurity concerns associated with synthetic biology, such as the potential malevolent misuse of the technology for bioterrorism, or the possibility of accidental or intentional release of a harmful engineered organism into the community by it yourself (DIY) synthetic biology users.4 While these implications of synthetic biology are of great importance, our focus here is different. We address regulatory and oversight concerns and challenges, and provide recommended strategies for dealing with the potential risks to human health and the environment from the purposeful, beneficial application of synthetic biology. Private companies, universities, and other entities are fast developing numerous legitimate uses of synthetic biology, in areas such as energy production, chemical synthesis, and bioremediation.5 These anticipated uses do not come without risk, risk that is sometimes referred to as as opposed to bioterror. * * * * 6 7 To date, the capacity of the existing regulatory system to address these bioerror risks has received limited attention and investigation, particularly in the legal literature.Our analysis reveals that although the extant regulatory system is capable of sufficiently handling several aspects of these novel synthetic biology organisms, there are also a number of potentially troubling regulatory gaps. These gaps arise because synthetic biology presents particular challenges for the existing U.S. regulatory regime due to three atypical characteristics of this nascent technology: (1) synthetic biology organisms can evolve;7 (2) the traditional assumed relationship between mass and risk may break down for synthetic biology products;8 * and (3) the conventional regulatory focus of existing statutes on end-product chemicals may be a poor match in certain instances for a technology that produces novel organisms, with their own attendant risks, that, in turn, produce the end-product chemicals.9This Article begins in Part I with an overview of synthetic biology and an examination of the potential benefits and risks of expected early synthetic biology products. The Toxic Substances Control Act (TSCA) is anticipated to be the most significant pre-existing regulatory authority concerning potential human health and environmental impacts of synthetic biology. Part II will examine how well TSCA is suited for this role. Part III discusses a number of other statutes, including laws pertaining to hazardous waste, endangered species, and pesticides, as well as National Institutes of Health guidelines that will also play a role in synthetic biology management.10 Part IV introduces several innovative governance approaches that could shore up some of the gaps in the existing regulatory framework for synthetic biology. …

Recent Patents on Oligonucleotide Synthesis and Gene Synthesis

Gene synthesis is an emerging field which has widespread implications in synthetic biology and molecular biology. The field is constantly evolving which has led to key advances in oligonucleotide synthesis and gene synthesis technologies, with simplicity, cost effectiveness and high throughput. The miniaturization, multiplexing, microfluidic processing and the integrated microchip engineering will drive down cost and increase productivity without compromising DNA synthesis fidelity, whereas the gigantic amount of genome information provides infinite source of DNA elements and genes as raw material for synthetic biology. This article describes some of the recent patents on oligonucleotide synthesis and gene synthesis.

Research on small genomes: implications for synthetic biology

Synthetic genomics is a new field of research in which small DNA pieces are assembled in a series of steps into whole genomes. The highly reduced genomes of host-associated bacteria are now being used as models for de novo synthesis of small genomes in the laboratory. Bacteria with the smallest genomes identified in nature provide nutrients to their hosts, such as amino acids, co-factors and vitamins. Comparative genomics of these bacteria enables predictions to be made about the gene sets required for core cellular functions and the associated metabolic network for the biosynthesis of host-selected compounds. Synthetic biology may ultimately enable researchers to make customized cell-specific organelles for the production and delivery of drugs to humans and domestic animals. Synthetic genomics may also become the method of choice for functional analyses of genes and genomes from bacteria that cannot be cultivated in the laboratory.

A Synthetic Protein Selected for Ligand Binding Affinity Mediates ATP Hydrolysis

How primitive enzymes emerged from a primordial pool remains a fundamental unanswered question with important practical implications in synthetic biology. Here we show that a de novo evolved ATP binding protein, selected solely on the basis of its ability to bind ATP, mediates the regiospecific hydrolysis of ATP to ADP when crystallized with 1 equiv of ATP. Structural insights into this reaction were obtained by growing protein crystals under saturating ATP conditions. The resulting crystal structure refined to 1.8 A resolution reveals that this man-made protein binds ATP in an unusual bent conformation that is metal-independent and held in place by a key bridging water molecule. Removal of this interaction using a null mutant results in a variant that binds ATP in a normal linear geometry and is incapable of ATP hydrolysis. Biochemical analysis, including high-resolution mass spectrometry performed on dissolved protein crystals, confirms that the reaction is accelerated in the crystalline environment. This observation suggests that proteins with weak chemical reactivity can emerge from high affinity ligand binding sites and that constrained ligand-binding geometries could have helped to facilitate the emergence of early protein enzymes.

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.