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The limits of synthetic biology
Abstract
The pioneering works of Watson, Crick, Wilkins, and Franklin [1,2] on the structure of DNA have captivated our imaginations for over half a century and continue to shape our future endeavors. The genetic code, a mystery for many years, was soon thereafter decoded by organic chemists employing organic synthesis of polynucleotides [3]. Ever since, the construction of DNA has been central to our ability to probe the molecular nature of life. Synthetic biologists now push the limits of what can be engineered using DNA – from scratch if needed: complex genetic circuits, large metabolic pathways, and even whole genomes.
... It aims at creating "orthogonal" systems which should enjoy the homeostatic conditions of cells while pursuing their artificial functions in an uncorrelated manner with the rest of their biological environment. This approach has proven fecund, although sometimes its limitations where overlooked (51). In any case, it both managed to accomplish this orthogonality until some point and to provide very useful tools to act on biological systems. ...
... compensate or at least account for bleaching, it is necessary to measure the rate of fluorescence decay due to bleaching. The classic experiment consists in placing cells expressing the FP in similar conditions as the experiment 51 and leave excitation light shining on them while recording the diminution in fluorescence (106). ...
... This mild deviation from a pure exponential was found to be more pronounced at very low fluorescence level in our system (~300 AU). 51 In fact, beaching rate can be specific to a given strain in particular experimental conditions. 52 yECitrine, excited for 200ms every 5s at 50% intensity on an X--Cite 120PC lamp, under a 100X objective PlanApo 1.4 NA, Olympus 53 This means that with the sampling of 6 min rate used in chapter III, fluorescence decay from photobleaching has a rate of 5.3.10 --4 which is ten times less than protein dilution and is therefore negligible. ...
When shifted to a stressful environment, cells are capable of complex response and adaptations. Although the cellular response to a single stress has been studied in great detail, very little is known when it comes to dynamically fluctuating stressful environments. In addition, in the context of stress response, the role of cell-to-cell variability in cellular processes and more specifically in gene expression is still unclear. In this work, we use a systems and synthetic biology approach to investigate osmotic stress in S. cerevisiae at the single cell level. Combining microfluidics, fluorescent microscopy and advanced image analysis, we are able to subject cells to precise fluctuating osmolarity and monitor single-cell temporal response. While much previous research in gene expression heterogeneity focused on its stochastic aspect, we consider here long-lasting differences between cells regarding expression kinetics. Using population models and state-of-the-art statistical analysis, we manage to represent both population and single-cell dynamics in a single concise modelling framework. This quantitative approach capturing stable individuality in gene expression dynamics can define a form of non-genetic cellular identity. To improve our comprehension of the biological interpretation of such identity, we investigate the relation between single-cell specificities in their gene expression with their phenotype and micro-environment. We then take a lineage based perspective and find this form of identity to be partially inherited. Understanding the evolutionary consequences of inheritable non-genetic cellular identity requires a better knowledge of the impact of fluctuating stress on cell proliferation. Dissecting quantitatively the consequences of repeated stress on cell-cycle and growth gives us an overview of the energetic and temporal consequences of repeated stress. At last, technical and theoretical developments needed to carry this investigation further are presented. These include the use of automated experimental design, both offline and online through real-time experimental design and single-cell real-time control of gene expression.
... Synthetic biology has aimed to systematize the engineering of microbial systems with a dual aim: understanding the principles by which living organisms process information, and constructing synthetic biosystems with enhanced (or even novel) traits for concrete biotechnological applications (Khalil and Collins, 2010;Leonard et al., 2008). While progress has been made over recent years, the latter objective has remained elusive (Hanson and Lorenzo, 2023;Zakeri and Carr, 2015). ...
... While the basic idea is appealing, we must consider its practical limitations if the end goal is to develop microbial communities for biotechnological purposes which can be deployed on a large scale. Over the past decades, synthetic biologists have devoted much effort to addressing the challenges of engineering single organisms (e.g., Perrino et al., 2021;Müller et al., 2019;Slusarczyk et al., 2012), with only limited practical success (Hanson and Lorenzo, 2023;Zakeri and Carr, 2015). As the field of synthetic ecology develops, we must carefully assess the risk of falling into a similar stage of stagnation. ...
Microbial communities are able to carry out myriad functions of biotechnological interest, ranging from the degradation of industrial waste to the synthesis of valuable chemical products. Over the past years, several strategies have emerged for the design of microbial communities and the optimization of their functions. Here we provide an accessible overview of these strategies. We highlight how principles of synthetic biology, originally devised for the engineering of individual organisms and sub-organismal units (e.g., enzymes), have influenced the development of the field of synthetic microbial ecology. With this, we aim to encourage readers to critically evaluate how insights from synthetic biology should guide our approach to community-level engineering.
... On the other hand, challenges such as the complexity of designed genetic circuits, great metabolic pathways, thermodynamic limits of manufacturing molecules in the biological environment, and limits of evolution should be identified. Nevertheless, there are many opportunities to minimize the negative effects of evolution on our designs [67]. ...
Synthetic biology breakthroughs have facilitated genetic circuit engineering to program cells through novel biological functions, dynamic gene expressions, as well as logic controls. SynBio can also participate in the rapid development of new treatments required for the human lifestyle. Moreover, these technologies are applied in the development of innovative therapeutic, diagnostic, as well as discovery-related methods within a wide range of cellular and molecular applications. In the present review study, SynBio applications in various cellular and molecular fields such as novel strategies for cancer therapy, biosensing, metabolic engineering, protein engineering, and tissue engineering were highlighted and summarized. The major safety and regulatory concerns about synthetic biology will be the environmental release, legal concerns, and risks of the engineered organisms. The final sections focused on limitations to SynBio.
... For example, benefit from the lower cross-reactivity and higher sensitivity of engineered parts, independent control of gene expression by 12 inducers could be realized in one cell (Meyer et al., 2019). However, it is still challenging to design reliable devices and circuits because the behaviors of the biological components that regulate the complex circuits are still poorly predictable in the chassis (Zakeri and Carr, 2015;Zhang et al., 2016). Engineered biological components with better performance are expected to decrease the uncertainty of synthetic systems. ...
Precise regulation of gene expression is fundamental for tailor-made gene circuit design in synthetic biology. Current strategies for this type of development are mainly based on directed evolution beginning with a native promoter template. The performances of engineered promoters are usually limited by the growth phase because only one promoter is recognized by one type of sigma factor (σ). Here, we constructed multiple-σ recognizable artificial hybrid promoters (AHPs) composed of tandems of dual and triple natural minimal promoters (NMPs). These NMPs, which use σA, σH and σW, had stable functions in different growth phases. The functions of these NMPs resulted from an effect called transcription compensation, in which AHPs sequentially use one type of σ in the corresponding growth phase. The strength of the AHPs was influenced by the combinatorial order of each NMP and the length of the spacers between the NMPs. More importantly, the output of the precise regulation was achieved by equipping AHPs with synthetic ribosome binding sites and by redesigning them for induced systems. This strategy might offer promising applications to rationally design robust synthetic promoters in diverse chassis to spur the construction of more complex gene circuits, which will further the development of synthetic biology.
... The last challenge to be addressed is the lack of tools for controlling the evolution of bacterial chassis -see (Bull and Barrick, 2017;Ellis, 2019;Nørholm, 2018;Zakeri and Carr, 2015) for a more comprehensive view on the topic, which might always hinder the efficiency of novel molecular toolkits in unexpected ways, such as through the generation of spontaneous point-mutations and intra-plasmid recombination (Oliveira et al., 2009) or through the deleterious action of endogenous transposable elements in the engineered/bio-production system (Geng et al., 2019;Oliveira et al., 2009;Rugbjerg et al., 2018). Up to now, most of the information regarding chassis evolution has been generated for model organisms such as E. coli, however they depict a phenomenon that permeates all biological systems, imposing great constraints in the development of novel molecular tools for non-model organisms. ...
A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.
... The challenge of defining biological robustness is difficult, because zero 44 evolution corresponds to maximal genetic robustness, but may be most efficiently 45 implemented by aiming for zero fitness, i.e. maximal fragility upon environmental 1 release; fragility, in turn, should be implemented in a robust way (e.g., as a reliable 2 safety lock), to avoid evolutionary escape. Moreover, evolution is an inherent feature of 3 living systems, determined by the fundamental property of error-prone self-replication, 4 even though evolutionary rates may potentially be reduced in engineered systems 5 (Zakeri and Carr, 2015). 6 "In the end, safety is decided by humans" (Fischhoff et al., 1978) and an acceptable 7 level of risk must be assessed based on agreed thresholds using data generated from 8 agreed protocols and metrics, and interpreted in the context of socioeconomic 9 ...
In Opinion I on Synthetic Biology (SynBio), the three Scientific Committees SCHER, SCENIHR and SCCS answered three questions from the European Commission on the scope, definition and identification of the relationship between SynBio and genetic engineering and the possibility of distinguishing the two. The definition reads: Synthetic Biology is the application of science, technology and engineering to facilitate and accelerate the design, manufacture and/or modification of genetic materials in living organisms. In Opinion II, the three Scientific Committees addressed five questions focused on the implications of likely developments in SynBio for humans, animals and the environment and on determining whether existing health and environmental risk assessment practices of the European Union for Genetically Modified Organisms are adequate for SynBio. Additionally, the Scientific Committees were asked to provide suggestions for revised risk assessment methods and risk mitigation procedures including safety locks. The current Opinion addresses specific risks to the environment from SynBio organisms, processes and products, partly in the context of Decision XI/11 of the Convention of Biodiversity (CBD) (CBD)(CBD)(CBD), identifies major gaps in knowledge to be considered for performing a reliable risk assessment and provides research recommendations resulting from gaps identified. The Scientific Committees confined the scope of their analysis to the foreseeable future, acknowledging that its findings should be reviewed and updated again after several years, depending on the development of the SynBio technology. Outside the scope of the current mandates are specific, thorough analyses of social, governance, ethical and security implications as well as human embryonic research.
Living systems are composed of a complex network of bioactive small molecules, proteins, ions and electrons, which present a wealth of opportunities for researchers to explore. Recently, organic chemists have developed a keen interest to move chemical reactions from laboratory flasks into living systems. This offers a new avenue for addressing challenges in current organic chemistry, expands the range of chemical transformations accessible to living systems, and provides a versatile tool for understanding and manipulating living systems. In this tutorial review, we include both the fundamental mechanisms and specific examples of how chemical reactions that typically occur outside of the biological context can be adapted for use within living systems. We also highlight the use of the resulting functional organic materials for biomedical applications including but not limited to imaging, therapy, theranostics and organic electronics. Finally, we discuss current challenges and future perspectives in this exciting field. We envision this tutorial review will serve as a guide for designing new chemical reactions and pathways in living systems that can expand the range of biological processes and functions and will accelerate the development of new biomaterials, biocatalysts, and therapeutics for precision medicine and other social needs.
Mycoplasmas have exceptionally streamlined genomes and are strongly adapted to their many hosts, which provide them with essential nutrients. Owing to their relative genomic simplicity, Mycoplasmas have been used to develop chassis for biotechnological applications. However, the dearth of robust and precise toolkits for genomic manipulation and tight regulation has hindered any substantial advance. Herein we describe the construction of a robust genetic toolkit for M. pneumoniae, and its successful deployment to engineer synthetic gene switches that control and limit Mycoplasma growth, for biosafety containment applications. We found these synthetic gene circuits to be stable and robust in the long-term, in the context of a minimal cell. With this work, we lay a foundation to develop viable and robust biosafety systems to exploit a synthetic Mycoplasma chassis for live attenuated vectors for therapeutic applications. Mycoplasmas are minimal cell model organisms but lack genetic tools. Here the authors provide a robust genetic toolkit for Mycoplasma demonstrating gene circuit engineering applications.
Mycoplasmas have exceptionally streamlined genomes and are strongly adapted to their many hosts, which provide them with essential nutrients. Owing to their relative genomic simplicity, Mycoplasmas have been used for the development of chassis to deploy tailored vaccines. However, the dearth of robust and precise toolkits for genomic manipulation and tight regulation has hindered any substantial advance. Herein we describe the construction of a robust genetic toolkit for M. pneumoniae , and its successful deployment to engineer synthetic gene switches that control and limit Mycoplasma growth, for biosafety containment applications. We found these synthetic gene circuits to be stable and robust in the long-term, in the context of a minimal cell. With this work, we lay a foundation to develop viable and robust biosafety systems to exploit a synthetic Mycoplasma chassis for live attenuated vaccines or even for live vectors for biotherapeutics.
Much effort has been expended on building cellular computational devices for different applications. Despite the significant advances, there are still several addressable restraints to achieve the necessary technological transference. These improvements will ease the development of end-user applications working out of the lab. In this study, we propose a methodology for the construction of printable cellular devices, digital or analogue, for different purposes. These printable devices are designed to work in a 2D surface, in which the circuit information is encoded in the concentration of a biological signal, the so-called carrying signal. This signal diffuses through the 2D surface and thereby interacts with different device components. These components are distributed in a specific spatial arrangement and perform the computation by modulating the level of the carrying signal in response to external inputs, determining the final output. For experimental validation, 2D cellular circuits are printed on a paper surface by using a set of cellular inks. As a proof-of-principle, we have printed and analysed both digital and analogue circuits using the same set of cellular inks but with different spatial topologies. The proposed methodology can open the door to a feasible and reliable industrial production of cellular circuits for multiple applications.
This article discusses the contingencies and complexities of CRISPR. It outlines key problems regarding off-target effects and replication of experimental work that are important to consider in light of CRISPR’s touted ease of use and diffusion. In light of literature on the sociotechnical dimensions of the life sciences and biotechnology and literature on former bioweapons programs, this article argues that we need more detailed empirical case studies of the social and technical factors shaping CRISPR and related gene-editing techniques in order to better understand how they may be different from other advances in biotechnology — or whether similar features remain. This information will be critical to better inform intelligence practitioners and policymakers about the security implications of new gene-editing techniques.
Recent work in developing self-replicating machines has approached the problem as an engineering problem, using engineering materials and methods to implement an engineering analogue of a hitherto uniquely biological function. The question is – can anything be learned that might be relevant to an astrobiological context in which the problem is to determine the general form of biology independent of the Earth. Compared with other non-terrestrial biology disciplines, engineered life is more demanding. Engineering a self-replicating machine tackles real environments unlike artificial life which avoids the problem of physical instantiation altogether by examining software models. Engineering a self-replicating machine is also more demanding than synthetic biology as no library of functional components exists. Everything must be constructed de novo . Biological systems already have the capacity to self-replicate but no engineered machine has yet been constructed with the same ability – this is our primary goal. On the basis of the von Neumann analysis of self-replication, self-replication is a by-product of universal construction capability – a universal constructor is a machine that can construct anything (in a functional sense) given the appropriate instructions (DNA/RNA), energy (ATP) and materials (food). In the biological cell, the universal construction mechanism is the ribosome. The ribosome is a biological assembly line for constructing proteins while DNA constitutes a design specification. For a photoautotroph, the energy source is ambient and the food is inorganic. We submit that engineering a self-replicating machine opens up new areas of astrobiology to be explored in the limits of life.
Synthetic biology offers a powerful method to design and construct biological devices for human purposes. Two prominent design methodologies are currently used. Rational design adapts the design methodology of traditional engineering sciences, such as mechanical engineering. Directed evolution, in contrast, models its design principles after natural evolution, as it attempts to design and improve systems by guiding them to evolve in a certain direction. Previous work has argued that the primary difference between these two is the way they treat variation: rational design attempts to suppress it, whilst direct evolution utilizes variation. I argue that this contrast is too simplistic, as it fails to distinguish different types of variation and different phases of design in synthetic biology. I outline three types of variation and show how they influence the construction of synthetic biological systems during the design process. Viewing the two design approaches with these more fine-grained distinctions provides a better understanding of the methodological differences and respective benefits of rational design and directed evolution, and clarifies the constraints and choices that the different design approaches must deal with.
After tackling the genomes of bacteria and yeast, synthetic biologists are setting their sights on rewriting those of more complex organisms, including humans.
Este artículo aborda críticamente 1) los aspectos especificamente tecnocientíficos de la biología sintética, 2) la función de las promesas biotécnicas y biopolíticas de perfectibilidad de la «vida en sí»1, y 3) el problemático concepto de «biología digital». La biología sintética rechaza la idea de una naturaleza dada: la «vida en sí» se define como un ámbito de potencialidades, con materiales adaptables y estructuras flexibles que pueden utilizarse para rediseñar y «perfeccionar» la naturaleza. Los bioingenieros dicen crear organismos vivos desde cero, utilizando componentes genéticamente estandarizados y diseños informáticos: estas «máquinas vivas», que no existen en la naturaleza, están supuestamente al servicio de fines humanos. Yendo más allá del actual (y limitado) estado de las investigaciones, algunas voces dentro de la biología sintética hacen declaraciones osadas sobre escenarios socio-técnicos, objetos imaginarios y experimentos biotecnológicos para el futuro. Éstos no se desarrollarían tras las puertas de los laboratorios, sino más bien en el seno de la sociedad. Con sus visiones, los biólogos sintéticos se están transformando en los ingenieros de las sociedades del futuro. La biología sintética, por tanto, promueve una «biotecnologización del futuro colectivo», además de formar parte de una «economía de la promesa» tecnocientífica que aspira a colonizar el futuro -lo que nos obliga a repensar la cuestión foucaultiana de la biopolítica. Para la promesa de una «biología digital», lanzada por la biología sintética, es vital la existencia de imaginarios centrados en el código, biocibernéticos e incluso transhumanistas. Éstos impulsan perspectivas que entienden la «vida y la naturaleza» como un ámbito de riquezas potenciales, incluso ilimitadas, que pueden programarse y producirse mediante procedimientos informáticos: «escribir» el código de la vida.
This chapter engages with (1) synthetic biology’s technoscientific specifica, (2) the role of promises, and (3) the problematic notion of ‘digital biology’. Synthetic biology dismisses the idea of an already given nature: ‘life itself’ is conceptualized as a field of potentialities, with adaptable materials and flexible structures that can be used for re-engineering to ‘perfect’ nature. Bioengineers claim to create new living organisms from scratch, using genetically standardized parts and computer-based design: ‘Living machines’ which do not exist in nature are supposed to serve human purposes. Beyond its actual (and limited) state of research, some voices of synthetic biology offer bold claims of socio-technical scenarios, imagined objects, and future biotechnical experiments, which take place in society rather than behind laboratory doors. With their visions, synthetic biologists are becoming engineers of future societies. Synthetic biology develops a ‘biotechnologization of collective futures’ and it is part of a technoscientific ‘promise-economy’ that aims to colonize the future. Crucial for synthetic biology’s promise of ‘digital biology’ are script-centered, bio-cybernetic, and even transhumanist figures of thought that fuel new visions of life and nature as a field of potentials and even limitless treasures that can be programmed and produced by computational procedures: ‘writing’ the code of life.
Protein-protein interactions are fundamental to many biological processes. Yet the weak and transient non-covalent bonds that characterize most protein-protein interactions found in nature impose limits on many bioengineering experiments. Here a new class of genetically encodable peptide-protein pairs-isopeptag-N/pilin-N, isopeptag/pilin-C, and SpyTag-SpyCatcher-that interact via autocatalytic intermolecular isopeptide bond formation is described. Reactions between peptide-protein pairs are specific, robust, orthogonal, and able to proceed under most biologically relevant conditions both in vitro and in vivo. As fusion constructs they provide a handle on molecules of interest, both organic and inorganic, that can be grasped with an iron grip. Such stable interactions provide robust post-translational control over biological processes, and open new opportunities in synthetic biology for engineering programmable and self-assembling protein nanoarchitectures.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
How do scientists perceive the media coverage of synthetic biology (SB)? In this paper, we approach this question by studying a set of cartoons devoted to SB. Based on a categorization of the cartoons into five large thematic groups an international survey was carried out to assess the opinion of SB research groups on science communication with regard to the public image of their discipline. The 101 responses obtained indicate that in general, their perception of the communication is not negative, although many respondents raised concerns on the media’s inclination to sensationalism and over-simplification. However, the results also suggest that (in the light of the unfortunate experiences with GMO communication) scientists should think twice before proposing metaphorical interpretations of their research.
Francis Crick reviews the papers published 21 years ago on the structure of DNA and the reaction to them.
The DNA-like polymers, poly d-TC:AG and poly d-TG:AC, which contain in each strand only two bases in alternating sequence, have been used as templates for DNA-dependent RNA polymerase. In this way, four single-stranded long-chain ribopolynucleotides, poly UC, poly AG, poly UG and poly AC containing in every case the two nucleotides in alternating sequence have been prepared. The messenger activity of poly UC in the Escherichia coli cell-free amino acid incorporation system has been studied. The ribopolynucleotide stimulates the incorporation of only two amino acids, serine and leucine, in equimolar amounts. The product of the reaction has been characterized as a co-polypeptide containing serine and leucine in alternating sequence.