A synthetic synthesis to explore animal evolution and development.

Does evolution proceed in small steps or large leaps? How repeatable is evolution? How constrained is the evolutionary process? Answering these long-standing questions in evolutionary biology is indispensable for both understanding how extant biodiversity has evolved and predicting how organisms and ecosystems will respond to changing environments in the future. Understanding the genetic basis of phenotypic diversification and speciation in natural populations is key to properly answering these questions. The leap forward in genome sequencing technologies has made it increasingly easier to not only investigate the genetic architecture but also identify the variant sites underlying adaptation and speciation in natural populations. Furthermore, recent advances in genome editing technologies are making it possible to investigate the functions of each candidate gene in organisms from natural populations. In this article, we discuss how these recent technological advances enable the analysis of causative genes and mutations and how such analysis can help answer long-standing evolutionary biology questions.This article is part of the theme issue ‘Genetic basis of adaptation and speciation: from loci to causative mutations’.

Diamondback terrapins (Malaclemys terrapin centrata) exhibit strong environmental adaptability and live in both freshwater and saltwater. However, the genetic basis of this adaptability has not been the focus of research. In this study, we successfully constructed a ∼2.21-Gb chromosome-level genome assembly for M. t. centrata using high-coverage and high-depth genomic sequencing data generated on multiple platforms. The M. t. centrata genome contains 25 chromosomes and the scaffold N50 of ∼143.75 Mb, demonstrating high continuity and accuracy. In total, 53.82% of the genome assembly was composed of repetitive sequences, and 22435 protein-coding genes were predicted. Our phylogenetic analysis indicated that M. t. centrata was closely related to the red-eared slider turtle (Trachemys scripta elegans), with divergence approximately ∼23.6 million years ago (Mya) during the early Neogene period of the Cenozoic era. The population size of M. t. centrata decreased significantly over the past ∼14 Mya during the Cenozoic era. Comparative genomic analysis indicated that 36 gene families related to ion transport were expanded and several genes (AQP3, solute carrier subfamily, and potassium channel genes) underwent specific amino acid site mutations in the M. t. centrata genome. Changes to these ion transport-related genes may have contributed to the remarkable salinity adaptability of diamondback terrapin. The results of this study not only provide a high-quality reference genome for M. t. centrata but also elucidate the possible genetic basis for salinity adaptation in this species.

Context-aware synthetic biology by controller design: Engineering the mammalian cell.

The application of synthetic biology approaches to study development opens the possibility to build and manipulate developmental processes to understand them better. Researchers have reconstituted fundamental developmental processes, such as cell patterning and sorting, by engineering gene circuits in vitro. Moreover, new tools have been created that allow for the control of developmental processes in more complex organoids and embryos. Synthetic approaches allow testing of which components are sufficient to reproduce a developmental process and under which conditions as well as what effect perturbations have on other processes. We envision that the future of synthetic developmental biology requires an increase in the diversity of available tools and further efforts to combine multiple developmental processes into one system.

Applications of Synthetic Biology in Microbial Biotechnology

The Centre for Synthetic and Systems Biology ('SynthSys') was originally established in 2007 as the Centre for Integrative Systems Biology, funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the Engineering and Physical Sciences Research Council (EPSRC). Today, SynthSys embraces an extensive multidisciplinary community of more than 200 researchers from across the University with a common interest in synthetic and systems biology. Our research is broad and deep, addressing a diversity of scientific questions, with wide ranging impact. We bring together the power of synthetic biology and systems approaches to focus on three core thematic areas: industrial biotechnology, agriculture and the environment, and medicine and healthcare. In October 2015, we opened a newly refurbished building as a physical hub for our new U.K. Centre for Mammalian Synthetic Biology funded by the BBSRC/EPSRC/MRC as part of the U.K. Research Councils' Synthetic Biology for Growth programme.

Editorial: Synthetic biology--ready for application.

Filamentous fungi are frequently used as hosts for heterologous production of industrial enzymes and bioactive secondary metabolites. This role will likely increase in the post-genomic era as a multitude of fungal genome sequences are mined for new valuable products. The trend for constructing fungal cell factories is to use synthetic biology approaches. In simplicity, this includes puzzling available bio-blocks together using seamless DNA fusions to generate vectors, gene-expression cassettes, and protein fusions. Moreover, it also sets the stage to expand natural enzymatic functions and chemistry by combining functional domains into synthetic proteins with novel enzymatic properties or by combining enzymes from different organisms in a common host to establish new biosynthetic pathways. In this chapter, we describe the components of a modern synthetic biology toolbox and how they have been used to construct and optimize fungal cell factories for heterologous production of natural and synthetic enzymes and secondary metabolites.

Characterizing biological pathways at the genome scale is one of the most important and challenging tasks in the post genomic era. To address this challenge, we have developed a computational method to systematically and automatically derive partial biological pathways in yeast using high-throughput biological data, including yeast two hybrid data, protein complexes identified from mass spectroscopy, genetics interactions, and microarray gene expression data in yeast Saccharomyces cerevisiae. The inputs of the method are the upstream starting protein (e.g., a sensor of a signal) and the downstream terminal protein (e.g., a transcriptional factor that induces genes to respond the signal); the output of the method is the protein interaction chain between the two proteins. The high-throughput data are coded into a graph of interaction network, where each node represents a protein. The weight of an edge between two nodes models the closeness of the two represented proteins in the interaction network and it is defined by a rule-based formula according to the high-throughput data and modified by the protein function classification and subcellular localization information. The protein interaction cascade pathway in vivo is predicted as the shortest path identified from the graph of the interaction network using Dijkstra's algorithm. We have also developed a web server of this method (http://compbio.ornl.gov/structure/pathway) for public use. To our knowledge, our method is the first automated method to generally construct partial biological pathways using a suite of high-throughput biological data. This work demonstrates the proof of principle using computational approaches for discoveries of biological pathways with high-throughput data and biological annotation data.

Cell Biology 2.0

Chapter 5 - Computational Design of Biological Systems: From Systems to Synthetic Biology

Johanna Schmitt It is a great pleasure to help honour the 2014 recipient of the Molecular Ecology Prize: Johanna Schmitt, Professor of Evolution and Ecology and of Population Biology at the University of California, Davis. Johanna, or Annie as she is known by friends and colleagues, has had tremendous influence on the field of ecological genetics throughout her career, and her recent work on the genetic basis of adaptation in Arabidopsis thaliana is some of the most ambitious applications of genomic methods to test hypotheses of ecological and evolutionary dynamics. Entering the field of evolutionary genetics and genomics from the field of ecology, she has infused genetic studies of adaptation with a rich and nuanced view of the ecological environment as seen from the perspective of her study organisms. Anyone who has walked in the woods with her will recognize her plants' eye view in her research. As her former postdoc John Stinchcombe observed, ‘one of the things I find remarkable about Annie (among many) is that as the field has transitioned from a few genes or anonymous markers to whole genome level variation, she's never lost her “feel for the organism” or sight of the larger ecological or evolutionary questions that motivated her to go down this path’. Annie majored in Biology at Swarthmore College and continued her PhD in Biology at Stanford University, with Ward Watt as her advisor. There, she wrote her dissertation on the pollination biology of Scenecio and Linanthus, cultivating interests in the population genetic consequences of density-dependent pollination dynamics (e.g. Schmitt 1983a,b). It was during her postdoctoral work at Duke University, with Janis Antonovics (whom she admired as a great female role model, until she met him in person), that she developed her signature methodology of applying genetic designs to clever and complex field experiments. This approach had two important consequences for her own research and for the field of ecological genetics: first, it illustrated how ecological manipulations can be combined with genetic analysis to test evolutionary hypotheses. For example, her work at Duke tested how genetic diversity within local neighbourhoods can influence competitive interactions and adverse effects of herbivores, relating these dynamics to the evolution of sexual reproduction (Schmitt & Antonovics 1986b; Schmitt & Ehrhardt 1987; Kelley et al. 1988). The focus on sexual reproduction also motivated her to distinguish maternal vs. paternal effects on progeny phenotypes, bringing into focus the phenomenon of maternal effects or cross-generational phenotypic plasticity (Antonovics & Schmitt 1986; Schmitt & Antonovics 1986a). Second, this approach illustrated the strong environmental context of the expression of genetically based traits. Her subsequent work, which she continued at her first faculty appointment at Brown University, engaged the evolutionary and ecological consequences of this environment-dependent genetic expression or genotype–environment interaction. She made phenotypic plasticity a central focus of her research programme (Schmitt et al. 1992; Schmitt 1993, 1995). It was this work that pioneered methods for testing the adaptive significance of phenotypic plasticity, both within and across generations (Schmitt 1993, 1997; Wulff et al. 1994, Schmitt et al. 1999). Her combination of environmental manipulations, phenotypic and genetic manipulations, and measurements of environment-dependent natural selection became the gold standard of tests for adaptive plasticity. Her work on shade avoidance responses in Impatiens capensis unambiguously demonstrated adaptive plasticity and documented that not only did phenotypes change in response to environmental conditions, but genetic variances and covariances did as well (Dudley & Schmitt 1996; Schmitt & Dudley 1996; Donohue et al. 2000a,b). That is, the genetic basis of traits under selection, and the genetic relationships among them, depended strongly on the ecological environment they experienced. Annie's work on shade avoidance responses engaged not only the quantitative genetic basis of this complex trait, but the molecular genetic pathways associated with it as well. Shade avoidance—the ability of plants to elongate in response to vegetation shade—was long known to be mediated by the plant photoreceptors, phytochromes (Schmitt & Wulff 1993). During a sabbatical at the University of Leicester, she collaborated with Alex McCormac and Harry Smith to test how the genetic disruption of phytochrome function would alter shade avoidance and fitness. Using transgenic lines of tobacco whose shade avoidance ability had been blocked, and constitutively shade-avoiding mutants of Brassica, they demonstrated a significant fitness disadvantage of inappropriate shade avoidance responses (Schmitt et al. 1995). This was her first work that employed tools of molecular genetics to test ecological hypotheses. While continuing to investigate the quantitative genetic basis of diverse plastic responses to vegetation shade, Annie began to explore other genetic methods to evaluate their genetic architecture. Like other evolutionary geneticists at the time, she discovered the utility of employing natural genetic variation in ecologically important traits to investigate their genetic basis through quantitative trait locus (QTL) mapping. At the time when QTL analysis was just beginning to be broadly applied to identify loci associated with ecologically significant phenotypes, Annie and her associates implemented a highly ambitious QTL study using Arabidopsis thaliana under field conditions to map not only well-defined phenological and morphological phenotypes, but fitness itself. This intense collaborative effort, initially supported by an NSF FIBR grant, was among the very first to map loci associated with fitness under natural conditions in contrasting geographical sites (Weinig et al. 2002, 2003a,b,c). By demonstrating that some genetic loci were associated with fitness only in one location but neutral in another, while other genetic loci were associated with fitness in both locations, but in opposite directions, this study illustrated how QTL analysis could be employed to resolve long-standing issues of trade-offs in adaptation across geographical locations—specifically revealing instances of conditional neutrality and evidence of antagonistic pleiotropy. The success of this research programme spawned a monster, according to the numerous participants of the next major research effort. Encouraged by the success of the two-site field study with numerous recombinant inbred lines, the team, with some new recruits, initiated a study using four sites across the native range of A. thaliana, from Oulu, Finland to Valencia, Spain, in which hundreds of natural ecotypes combined with a strategic array of mutants, were planted for continuous monitoring. Simultaneously with this ambitious field experiment, creative modelling efforts were being developed to predict the flowering time of specific genotypes under diverse climatic scenarios using agronomic models. This synthesis of genetics, ecology, agronomy and mathematical modelling was unique, and it provided unique insight into the genetic basis of adaptation. Their synthetic approach revealed, for example, that even well-known flowering time genes are expected to exhibit (and did exhibit) effects on flowering time only under certain ecological circumstances and life history backgrounds (Wilczek et al. 2009; Chew et al. 2012). The feat of bringing evolutionary ecologists (Cynthia Weinig, Tonia Korves, Amity Wilczek) in dialogue with population geneticists (Michael Purugganan), molecular geneticists (George Coupland, Rick Amasino, Caroline Dean) and modellers (Steve Welch), while engaging international collaborators (Outi Savolainen, Matthias Hoffmann) in fieldwork, was a Herculean accomplishment. A series of articles from this work was published in Science, PNAS, Molecular Ecology and a number of other prominent journals. Among the most notable findings of this programme were that A. thaliana shows evidence of climate adaptation, with geographic clines in adaptively significant life history traits as well as the loci associated with those traits (Caicedo et al. 2004; Stinchcombe et al. 2004, 2005; Korves et al. 2007; Wilczek et al. 2010). Moreover, genome-wide association studies revealed associations of loci with climate factors across the genome (Fournier-Level et al. 2011, 2013). Most recently (Wilczek et al. 2014), the team found evidence that climate change has caused banked seeds to no longer be optimally adapted to their locations of collection, but that that ecotypes from historically warmer locations performed better under current (warmer) conditions than banked seeds in their native location. As such, immigration of more warm-adapted genotypes into areas with climate change, not emergence from the seed bank or introduction of local banked seeds, is expected to be more effective at maintaining populations in the face of climate change. These empirical data, combined with predictive modelling, establishes a new standard for predictions of how organisms can respond to climate change. Annie was involved at the beginning stages of developing model genetic organisms into model ecological organisms. She helped shape the sorts of questions that could be addressed with this sort of collaboration and made ecological genetics a collaborative endeavour between ecologists, population geneticists and molecular geneticists. Always promoting collaboration over competition, she brokered many matches between PIs studying related phenomena and proposed opportunities to combine efforts in synergistic directions. The field has her to thank for the open and collaborative spirit she has infused it with. In addition to shaping the collaborative nature of the field of ecological genetics, Annie has been a valuable mentor to people at all stages. At Brown, she worked closely with her undergraduate students to involve them with every step of their research projects, from helping to design experiments to data collection, and analysis and presentation. Many of us are grateful for this effort, which has produced so many excellent students who have joined our laboratories as graduate students or technicians. It was here, too, that so many of her postdocs learned the craft of designing undergraduate projects that were self-contained, challenging and rewarding, providing a model for tapping the unique resources of undergraduates in research. Her numerous postdocs also benefitted from being members of such a cohesive laboratory, in which laboratory members could count on each other for technical help and conceptual exchange. Annie's generosity of time, creativity and opportunity were critical to the professional development of many of us. Personally, I will never take for granted the extreme generosity she extended to me when, after a very ill-timed postdoc in Yemen during what turned into its civil war, I found myself evacuated back stateside with no backup plan. I basically knocked on her door to ask for a short-term landing pad, and she opened it up in a manner I could never have expected. At that critical, awkward and very tricky time in my career, she welcomed me into her laboratory, involved me in the ongoing research and gave me new skills, intellectual companionship and a model for how to run a laboratory that was collegial, engaging and effective. I am certain that anyone who spent time in her laboratory benefitted in the same way, and several of her former postdocs (including Susan Dudley, Massimo Pigliucci, Cynthia Weinig, John Stinchcombe, Amity Wilczek and others) have expressed the same appreciation over the years. Annie has amassed several honours as a result of her creative contributions, including election to the National Academy of Sciences, the American Academy of Arts and Sciences, the American Association for the Advancement of Sciences and an Alexander von Humboldt Award, among others. She has been the President of the major professional societies in her field: the Society for the Study of Evolution and the American Society of Naturalists. While at Brown University, she was Stephen T. Olney Professor of Natural History, and she was also the director of the Environmental Change Initiative there, where she exercised her remarkable ability to communicate and synthesize across scientific subfields. UC Davis is now the beneficiary of Annie's energy and expertise, after she moved there in 2012. This Molecular Ecology Prize serves to honour her past accomplishments and inspire curiosity for what is to come.

Synthetic biology is an emerging field blending approaches and concepts derived from classic engineering disciplines with modern biological approaches. Concepts of modularity and orthogonality, i.e. the transfer of simple building blocks between unrelated chassis (host organisms), are guiding principles for the design and construction of artificial biological systems, which in their ultimate implementation can be artificial organisms. Synthetic biology is not only leading the way towards the engineering of useful organisms that serve human purposes, it is also a new way of approaching basic scientific questions to understand complex biological systems. The classic reductionist methodology by which scientists have dissected complex systems to understand their properties through understanding the functionality of isolated components, finds its counterpart in synthetic biology. If we can build complex biological processes, systems, and ultimately organisms from simple, fully understood functional modules using a set of defined rules, we must fully understand the system. At first this approach may sound almost naïve as with near certainty scientists will encounter spectacular 'failures' on the way to building complex biological systems. Undoubtedly, the result of synthetic biology efforts will be more than the sum of the individual components giving rise to complex systems with novel emergent properties, many of which are unexpected or even undesired. However, the process of learning from those 'failures' often through predictive modeling and simulation studies in parallel to the actual assembly and testing of artificial biological systems, will lead to novel insights into the function of complex biological systems in general. Plant and algal cells are complex with their extra organelle, the plastid, and are highly sophisticated in their metabolism enabling them to convert light, CO2 and minerals into the building blocks of cells, produce all oxygen in the atmosphere, thousands of specialized chemicals including drugs, and energy-rich compounds that fuel life on earth. While engineers have been dabbling for many years in the redesign of bacterial and yeast chassis with novel properties, the application of synthetic biology to photosynthetic organisms is just beginning. Therefore, it seems timely to provide an overview of the state of the art of 'Synthetic Biology for Basic and Applied Plant Research' in this special issue of The Plant Journal. Next Generation Sequencing has given us a nearly unlimited number of genomic blueprints for photosynthetic bacteria, algae and plants and this provides the raw material for synthetic biology. Tools for recombining of genes and introducing them into an increasing number of photosynthetic chassis including organelles such as chloroplasts, are available and no longer an impediment to the application of synthetic biology to plants. One revolutionary technique, the introduction of the CRISPR/CAS system for genome editing is now being applied to edit not only the plant genome, but also the transcriptome and epigenome as discussed by Puchta (2016). Bacterial microcompartments, first discovered as carboxysomes in cyanobacteria, provide an important platform for the engineering of synthetic modules. They can encapsulate enzymes, concentrate substrates, and help in the avoidance of toxic products as Gonzalez-Esquer et al. (2016) describe. Cyanobacteria address one key problem that all photosynthetic organisms encounter, the natural inefficiency of the carbon-fixing enzyme RubBisCO, by encapsulating this enzyme in carboxysomes, which increases the local concentration of CO2 around the enzyme. Plants do not have a carboxysome-based carbon concentration mechanism to overcome the limitation of photosynthesis through RubBisCO's inefficiency. The solution could be to introduce this bacterial microcompartment into chloroplasts of crop plants and synthetic biology efforts towards this aim are well under way as described by Hanson et al. (2016). A subset of plants has evolved their own way of overcoming this problem by prefixing carbon using a more efficient enzyme than RubBisCO. This carbon concentration mechanism requires the compartmentalization of different sets of enzymes in different cells of the leaf, and this overall approach is referred to as C4-syndrome of C4 plants, because the CO2 is first fixed into a four-carbon compound rather than the three-carbon compound produced first by RuBisCO in C3 plants. Some of the important crop plants that feed the world are C4 plants, such as maize, but many are not, including wheat and rice. The solution is to engineer C4 photosynthesis in a C3 chassis and as Schuler et al. (2016) describe, efforts are well underway by applying synthetic biology. Introduction of orthogonal biosynthetic pathways into photosynthetic organelles and bacteria to enhance their synthetic repertoires requires a deep knowledge of the regulation of photosynthesis, as the balance of ATP/and NADPH and the nature of the carbon sink are critical for the efficiency of photosynthesis. Nielson and coworkers describe how optimization of carbon flux and reductant are critical elements in engineering cyanobacteria and chloroplasts to sustainably produce novel chemicals (Nielsen et al., 2015). Plants are capable of making a seemingly unlimited number of specialized compounds to defend themselves against pathogens or herbivores and many of these compounds have been used by humans for thousands of years, e.g. as drugs. One particular compound class, the terpenoids, provides an example of the amazing natural combinatorial chemistry that plants are capable of. Applying synthetic biology principles of modularity and orthogonality, plant engineers are now capable of recombining different modules of terpenoid biosynthesis from different sources into new chassis to engineer plants that produce new-to-nature compounds as Arendt et al. (2016) describe. Another spectacular success in recombining modules of genes derived from different plants, algae, and fungi into a new chassis, the industrial crop Camelina, is the production of oils with a near natural composition of healthy oils found in fish as summarized by Haslam et al. (2016). With this accomplishment, important sustainability and human health questions can be addressed. These include improving the sustainability of the aquaculture industry for the production of fish rich in omega-3 oils with well-known health benefits when part of the human diet. Another example of addressing pressing problems for humankind is the generation of sustainable feed-stocks for energy production, independent of fossil fuels. For this reason, many scientists are currently pursuing the engineering of dedicated biofuel crops through the application of synthetic biology principles as summarized by Shih et al. (2016). Plant signaling pathways are highly interconnected and redundant, and hence often hard to dissect using the classical reductionistic approaches. Synthetic Biology offers a new way to explore individual signaling pathways by reassembling them bottom up from modules in non-interfering backgrounds of new chassis. Braguy and Zurbriggen (2016) describe this approach in detail. Ultimately, understanding how signaling pathways feed into programmable plant genetic circuits will be essential for the engineering of plants to be more efficient or to produce novel compounds. Medford and Prasad (2016) explain how genetic parts such as promoters and other regulatory elements can be tested and their assembly into genetic circuits simulated. The list of examples and approaches described in this special issue of The Plant Journal is comprehensive. Our intention is that this special issue will explain key principles and areas of plant synthetic biology to guide the reader and future contributors of The Plant Journal in embracing these approaches for both fundamental and applied plant science. Other areas of interest not covered here include synthetic consortia, the synthetic interaction of photosynthetic and heterotrophic organisms beyond naturally occurring symbioses. As we learn to understand how the microbiome affects plant growth, synthetic biology approaches may be key in learning more about these complex interactions, a topic that certainly falls with in the scope of The Plant Journal. With the expansion of the current field of plant synthetic biology, The Plant Journal welcomes the submission of basic research papers applying synthetic biology to further our understanding of the full biological complexity of photosynthetic organisms and their complex biotic and abiotic interaction with the environment.

This chapter contains sections titled: Setting Systems and Synthetic Biology in Context Formation of Intellectual Orthodoxy for the First Scientific–Industrial Revolution Quiet Preparations for a Revolution Toward a New Complex Systems Paradigm and Philosophy References

Synthetic developmental biology: build and control multicellular systems