Making Security Viral: Shifting Engineering Biology Culture and Publishing.

The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes.

Biological and Molecular Components for Genetically Engineering Biosensors in Plants.

Plants adapt to their changing environments by sensing and responding to physical, biological, and chemical stimuli. Due to their sessile lifestyles, plants experience a vast array of external stimuli and selectively perceive and respond to specific signals. By repurposing the logic circuitry and biological and molecular components used by plants in nature, genetically encoded plant-based biosensors (GEPBs) have been developed by directing signal recognition mechanisms into carefully assembled outcomes that are easily detected. GEPBs allow for in vivo monitoring of biological processes in plants to facilitate basic studies of plant growth and development. GEPBs are also useful for environmental monitoring, plant abiotic and biotic stress management, and accelerating design-build-test-learn cycles of plant bioengineering. With the advent of synthetic biology, biological and molecular components derived from alternate natural organisms (e.g., microbes) and/or de novo parts have been used to build GEPBs. In this review, we summarize the framework for engineering different types of GEPBs. We then highlight representative validated biological components for building plant-based biosensors, along with various applications of plant-based biosensors in basic and applied plant science research. Finally, we discuss challenges and strategies for the identification and design of biological components for plant-based biosensors.

Guiding Ethical Principles in Engineering Biology Research.

Engineering biology is being applied toward solving or mitigating some of the greatest challenges facing society. As with many other rapidly advancing technologies, the development of these powerful tools must be considered in the context of ethical uses for personal, societal, and/or environmental advancement. Researchers have a responsibility to consider the diverse outcomes that may result from the knowledge and innovation they contribute to the field. Together, we developed a Statement of Ethics in Engineering Biology Research to guide researchers as they incorporate the consideration of long-term ethical implications of their work into every phase of the research lifecycle. Herein, we present and contextualize this Statement of Ethics and its six guiding principles. Our goal is to facilitate ongoing reflection and collaboration among technical researchers, social scientists, policy makers, and other stakeholders to support best outcomes in engineering biology innovation and development.

Quantitative and Predictive Genetic Parts for Plant Synthetic Biology.

Plant synthetic biology aims to harness the natural abilities of plants and to turn them to new purposes. A primary goal of plant synthetic biology is to produce predictable and programmable genetic circuits from simple regulatory elements and well-characterized genetic components. The number of available DNA parts for plants is increasing, and the methods for rapid quantitative characterization are being developed, but the field of plant synthetic biology is still in its early stages. We here describe methods used to describe the quantitative properties of genetic components needed for plant synthetic biology. Once the quantitative properties and transfer function of a variety of genetic parts are known, computers can select the optimal components to assemble into functional devices, such as toggle switches and positive feedback circuits. However, while the variety of circuits and traits that can be put into plants are limitless, doing synthetic biology in plants poses unique challenges. Plants are composed of differentiated cells and tissues, each representing potentially unique regulatory or developmental contexts to introduced synthetic genetic circuits. Further, plants have evolved to be highly sensitive to environmental influences, such as light or temperature, any of which can affect the quantitative function of individual parts or whole circuits. Measuring the function of plant components within the context of a plant cell and, ideally, in a living plant, will be essential to using these components in gene circuits with predictable function. Mathematical modeling will be needed to account for the variety of contexts a genetic part will experience in different plant tissues or environments. With such understanding in hand, it may be possible to redesign plant traits to serve human and environmental needs.

An FT-IR and XPS spectroscopy dataset of Pinus ponderosa sporopollenin and related samples to elucidate sporopollenin structural features.

The ATR FT-IR spectra of Pinus ponderosa sporopollenin isolated from pollen spores by enzymatic digestion. Sporopollenin is also isolated by solvent extraction, followed by either acidolysis with phosphoric acid, and acetolysis is reported [1]. The FT-IR spectra are supplemented by XPS data of the isolated sporopollenin samples. The enzymatically isolated sporopollenin is subjected to a variety of chemical treatments and modifications, including alkaline hydrolysis, deuteration (by both D20 and methanol-d4), sodium cyanoborohydride reduction, hydrolysis by peracetic acid, bromination, acetylization with acetone and octanal, and acid-catalyzed ketal cleavage. The sporopollenin isolated by acidolysis and acetolysis are also subjected to alkaline hydrolysis. The sporopollenin samples are compared to a variety of model compounds representative of putative structural constituents and functional groups.

Detailed characterization of Pinus ponderosa sporopollenin by infrared spectroscopy.

In plant spores and pollen, sporopollenin occurs as a structural polymer with remarkable resistance to chemical degradation. This recalcitrant polymer is well-suited to analysis by non-destructive infrared spectroscopy. However, existing infrared characterization of sporopollenin has been limited in scope and occasionally contradictory. This study provides a comprehensive structural analysis of sporopollenin in the Pinus ponderosa pollen exine using infrared spectroscopy, including detailed band assignments, descriptions of chemical reactivity, and comparison to multiple reference substances. We observe that the infrared spectral characteristics of sporopollenin prepared by enzymatic digestion of the polysaccharide-based intine are largely consistent with a copolymer of aliphatic lipids and trans-4-hydroxycinnamic acid, without distinct contributions from α-pyrone or carotenoid substructures.

Plant Biosystems Design Research Roadmap 1.0.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.

Engineering synthetic regulatory circuits in plants.

Plant synthetic biology is a rapidly emerging field that aims to engineer genetic circuits to function in plants with the same reliability and precision as electronic circuits. These circuits can be used to program predictable plant behavior, producing novel traits to improve crop plant productivity, enable biosensors, and serve as platforms to synthesize chemicals and complex biomolecules. Herein we introduce the importance of developing orthogonal plant parts and the need for quantitative part characterization for mathematical modeling of complex circuits. In particular, transfer functions are important when designing electronic-like genetic controls such as toggle switches, positive/negative feedback loops, and Boolean logic gates. We then discuss potential constraints and challenges in synthetic regulatory circuit design and integration when using plants. Finally, we highlight current and potential plant synthetic regulatory circuit applications.

Computational design of environmental sensors for the potent opioid fentanyl.

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

Foundational and Translational Research Opportunities to Improve Plant Health.

Reader Comments | Submit a Comment The white paper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on nonsustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.

Advancing Crop Transformation in the Era of Genome Editing.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

Towards programmable plant genetic circuits.

Synthetic biology enables the construction of genetic circuits with predictable gene functions in plants. Detailed quantitative descriptions of the transfer function or input-output function for genetic parts (promoters, 5' and 3' untranslated regions, etc.) are collected. These data are then used in computational simulations to determine their robustness and desired properties, thereby enabling the best components to be selected for experimental testing in plants. In addition, the process forms an iterative workflow which allows vast improvement to validated elements with sub-optimal function. These processes enable computational functions such as digital logic in living plants and follow the pathway of technological advances which took us from vacuum tubes to cell phones.

Quantitative characterization of genetic parts and circuits for plant synthetic biology.

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

A general strategy to construct small molecule biosensors in eukaryotes.

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

Loads bias genetic and signaling switches in synthetic and natural systems.

Biological protein interactions networks such as signal transduction or gene transcription networks are often treated as modular, allowing motifs to be analyzed in isolation from the rest of the network. Modularity is also a key assumption in synthetic biology, where it is similarly expected that when network motifs are combined together, they do not lose their essential characteristics. However, the interactions that a network module has with downstream elements change the dynamical equations describing the upstream module and thus may change the dynamic and static properties of the upstream circuit even without explicit feedback. In this work we analyze the behavior of a ubiquitous motif in gene transcription and signal transduction circuits: the switch. We show that adding an additional downstream component to the simple genetic toggle switch changes its dynamical properties by changing the underlying potential energy landscape, and skewing it in favor of the unloaded side, and in some situations adding loads to the genetic switch can also abrogate bistable behavior. We find that an additional positive feedback motif found in naturally occurring toggle switches could tune the potential energy landscape in a desirable manner. We also analyze autocatalytic signal transduction switches and show that a ubiquitous positive feedback switch can lose its switch-like properties when connected to a downstream load. Our analysis underscores the necessity of incorporating the effects of downstream components when understanding the physics of biochemical network motifs, and raises the question as to how these effects are managed in real biological systems. This analysis is particularly important when scaling synthetic networks to more complex organisms.

Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease.

BACKGROUND: A systematic method for plant genome manipulation is a major aim of plant biotechnology. One approach to achieving this involves producing a double-strand DNA break at a genomic target site followed by the introduction or removal of DNA sequences by cellular DNA repair. Hence, a site-specific endonuclease capable of targeting double-strand breaks to unique locations in the plant genome is needed. RESULTS: We engineered and tested a synthetic homing endonuclease, PB1, derived from the I-CreI endonuclease of Chlamydomonas reinhardtii, which was re-designed to recognize and cleave a newly specified DNA sequence. We demonstrate that an activity-optimized version of the PB1 endonuclease, under the control of a heat-inducible promoter, is capable of targeting DNA breaks to an introduced PB1 recognition site in the genome of Arabidopsis thaliana. We further demonstrate that this engineered endonuclease can very efficiently excise unwanted transgenic DNA, such as an herbicide resistance marker, from the genome when the marker gene is flanked by PB1 recognition sites. Interestingly, under certain conditions the repair of the DNA junctions resulted in a conservative pairing of recognition half sites to remove the intervening DNA and reconstitute a single functional recognition site. CONCLUSION: These results establish parameters needed to use engineered homing endonucleases for the modification of endogenous loci in plant genomes.

Crosstalk between endogenous and synthetic components--synthetic signaling meets endogenous components.

Synthetic biology uses biological components to engineer new functionality in living organisms. We have used the tools of synthetic biology to engineer detector plants that can sense man-made chemicals, such as the explosive trinitrotoluene, and induce a response detectable by eye or instrumentation. A goal of this type of work is to make the designed system orthogonal, that is, able to function independently of systems in the host. In this review, the design and function of two partially synthetic signaling pathways for use in plants is discussed. We describe observed interactions (crosstalk) with endogenous signaling components. This crosstalk can be beneficial, allowing the creation of hybrid synthetic/endogenous signaling pathways, or detrimental, resulting in system noise and/or false positives. Current approaches in the field of synthetic biology applicable to the design of orthogonal signaling systems, including the design of synthetic components, partially synthetic systems that utilize crosstalk to signal through endogenous components, computational redesign of proteins, and the use of heterologous components, are discussed.

Developing a synthetic signal transduction system in plants.

One area of focus in the emerging field of plant synthetic biology is the manipulation of systems involved in sensing and response to environmental signals. Sensing and responding to signals, including ligands, typically involves biological signal transduction. Plants use a wide variety of signaling systems to sense and respond to their environment. One of these systems, a histidine kinase (HK) based signaling system, lends itself to manipulation using the tools of synthetic biology. Both plants and bacteria use HKs to relay signals, which in bacteria can involve as few as two proteins (two-component systems or TCS). HK proteins are evolutionarily conserved between plants and bacteria and plant HK components have been shown to be functional in bacteria. We found that this conservation also applies to bacterial HK components which can function in plants. This conservation of function led us to hypothesize that synthetic HK signaling components can be designed and rapidly tested in bacteria. These novel HK signaling components form the foundation for a synthetic signaling system in plants, but typically require modifications such as codon optimization and proper targeting to allow optimal function. We describe the process and methodology of producing a synthetic signal transduction system in plants. We discovered that the bacterial response regulator (RR) PhoB shows HK-dependent nuclear translocation in planta. Using this discovery, we engineered a partial synthetic pathway in which a synthetic promoter (PlantPho) is activated using a plant-adapted PhoB (PhoB-VP64) and the endogenous HK-based cytokinin signaling pathway. Building on this work, we adapted an input or sensing system based on bacterial chemotactic binding proteins and HKs, resulting in a complete eukaryotic signal transduction system. Input to our eukaryotic signal transduction system is provided by a periplasmic binding protein (PBP), ribose-binding protein (RBP). RBP interacts with the membrane-localized chemotactic receptor Trg. PBPs like RBP have been computationally redesigned to bind small ligands, such as the explosive 2,4,6-trinitrotoluene (TNT). A fusion between the chemotactic receptor Trg and the HK, PhoR, enables signal transduction via PhoB, which undergoes nuclear translocation in response to phosphorylation, resulting in transcriptional activation of an output gene under control of a synthetic plant promoter. Collectively, these components produce a novel ligand-responsive signal transduction system in plants and provide a means to engineer a eukaryotic synthetic signaling system.

Programmable ligand detection system in plants through a synthetic signal transduction pathway.

BACKGROUND: There is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable. METHODOLOGY/PRINCIPAL FINDINGS: We describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant. CONCLUSIONS/SIGNIFICANCE: Our system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents.