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Abstraction hierarchy to define biofoundry workflows and operations for interoperable synthetic biology research and applications

Lack of standardization in biofoundries limits the scalability and efficiency of synthetic biology research. Here, we propose an abstraction hierarchy that organizes biofoundry activities into four interoperable levels: Project, Service/Capability, Workflow, and Unit Operation, effectively streamlining the Design‑Build‑Test‑Learn (DBTL) cycle. This framework enables more modular, flexible, and automated experimental workflows. It improves communication between researchers and systems, supports reproducibility, and facilitates better integration of software tools and artificial intelligence. Our approach lays the foundation for a globally interoperable biofoundry network, advancing collaborative synthetic biology and accelerating innovation in response to scientific and societal challenges.

Kim, Haseong↗

A frugal CRISPR kit for equitable and accessible education in gene editing and synthetic biology

Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit’s utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.

59 BASIC BIOLOGICAL SCIENCES↗

SyPro Poplar: Improving Poplar Biomass Production under Abiotic Stress Conditions: an Integrated Omics, Bioinformatics, Synthetic Biology and Genetic Engineering Approach (Final Report)

SyPro Poplar: Improving Poplar Biomass Production under Abiotic Stress Conditions: an Integrated Omics, Bioinformatics, Synthetic Biology and Genetic Engineering Approach In the SyPro Poplar project, we aimed to integrate omics, bioinformatics, synthetic biology, and genetic engineering approaches to develop transgenic poplar trees with sustained photosynthetic activity and increased biomass production under individual and the simultaneous occurrence of water deficit, increased soil salinity, and elevated temperatures. Specifically, we intended to (i) study the functions of selected stress-responsive genes at tissue and cell type-specific levels, (ii) discover novel motifs and construct stress-responsive synthetic promoters, and (iii) use these promoters to drive the expression of genes shown to confer abiotic stress tolerance in a variety of crops and develop abiotic stress-tolerant poplars in a coordinated fashion.

09 BIOMASS FUELS↗

Microbiome engineering for sustainable agriculture: using synthetic biology to enhance nitrogen metabolism in plant-associated microbes

Plants benefit from symbiotic relationships with their microbiomes. Modifying these microbiomes to further promote plant growth and improve stress tolerance in crops is a promising strategy. However, such efforts have had limited success, perhaps because the original microbiomes quickly re-establish. Since the complex biological networks involved are little understood, progress through conventional means is time-consuming. Synthetic biology, with its practical successes in multiple industries, could speed up this research considerably. Some fascinating candidates for production by synthetic microbiomes are organic nitrogen metabolites and related pyridoxal-5'-phosphate-dependent enzymes, which have pivotal roles in microbe-microbe and plant-microbe interactions. This review summarizes recent studies of these metabolites and enzymes and discusses prospective synthetic biology platforms for sustainable agriculture.

59 BASIC BIOLOGICAL SCIENCES↗

Perspectives for self-driving labs in synthetic biology

Self-driving labs (SDLs) combine fully automated experiments and data collection with artificial intelligence (AI) and control algorithms that decide not only the set of parameters for the next experiment, but also potentially which scientific hypotheses to test. Taken to their ultimate expression, SDLs could usher a new paradigm of scientific research, where the world is probed, interpreted, and explained by machines for human benefit. Whereas there are functioning SDLs in the fields of chemistry and materials science, we contend that synthetic biology provides a unique opportunity since the genome provides a single, easily accessible, target for affecting the incredibly wide repertoire of biological cell behavior. Since they can provide large amounts of high-quality data, SDLs can be a platform for AI to develop approaches to systematically convert data into scientific knowledge systems. These knowledge systems can be used both to understand the biological world and to design bioengineered systems to fit a desired specification (inverse design). However, the level of investment required for the creation of biological SDLs is only warranted if directed towards solving difficult and enabling biological questions. Here, we discuss challenges and opportunities in creating SDLs for synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

59 BASIC BIOLOGICAL SCIENCES↗

High-throughput chemical imaging for optimizing biofuel synthesis using synthetic biology (Final Technical Report)

Fatty acids can be produced biosynthetically in microbes and these compounds can serve as precursors to biodiesels and other high value oleochemicals. However, progress on engineering fatty acid biosynthesis, and biofuel synthesis more generally, has been hindered by current quantification methods that are either indirect or not amenable to high-throughput or single-cell resolution screening. In this project, we assembled an interdisciplinary team with complimentary expertise in synthetic biology and microscopy, metabolic engineering, and chemical imaging to address these challenges. We used chemical imaging to directly measure lipid biosynthesis in Escherichia coli engineered to produce fatty acids, obtaining detailed single-cell resolution measurements. We deployed stimulated Raman scattering (SRS) microscopy in concert with multiplexed genome engineering and gene circuit design strategies from synthetic biology to optimize production of fatty acids. These results provided novel insight into cell-to-cell heterogeneity present in biofuel production strains. In addition, we introduced new chemical imaging methods which are label-free and do not require fluorescent reporters. These efforts were complemented by other studied developing foundational tools for regulation and control, which offer excellent potential for advancing researchers’ ability to rapidly design, build, and test strains for enhanced biofuel synthesis.

09 BIOMASS FUELS↗

Permeability-Engineered Compartmentalization Enables In Vitro Reconstitution of Sustained Synthetic Biology Systems

In nature, biological compartments such as cells rely on dynamically controlled permeability for matter exchange and complex cellular activities. Likewise, the ability to engineer compartment permeability is crucial for in vitro systems to gain sustainability, robustness, and complexity. However, rendering in vitro compartments such a capability is challenging. Here, a facile strategy is presented to build permeability-configurable compartments, and marked advantages of such compartmentalization are shown in reconstituting sustained synthetic biology systems in vitro. Through microfluidics, the strategy produces micrometer-sized layered microgels whose shell layer serves as a sieving structure for biomolecules and particles. In this configuration, the transport of DNAs, proteins, and bacteriophages across the compartments can be controlled an guided by a physical model. Through permeability engineering, a compartmentalized cell-free protein synthesis system sustains multicycle protein production; ≈100 000 compartments are repeatedly used in a five-cycle synthesis, featuring a yield of 2.2 mg mL -1 . Further, the engineered bacteria-enclosing compartments possess near-perfect phage resistance and enhanced environmental fitness. In a complex river silt environment, compartmentalized whole-cell biosensors show maintained activity throughout the 32 h pollutant monitoring. It is anticipated that permeability-engineered compartmentalization should pave the way for practical synthetic biology applications such as green bioproduction, environmental sensing, and bacteria-based therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗

Enabling AI in synthetic biology through Construction File specification

The Construction File (CF) specification establishes a standardized interface for molecular biology operations, laying a foundation for automation and enhanced efficiency in experiment design. It is implemented across three distinct software projects: PyDNA_CF_Simulator, a Python project featuring a ChatGPT plugin for interactive parsing and simulating experiments; ConstructionFileSimulator, a field-tested Java project that showcases 'Experiment' objects expressed as flat files; and C6-Tools, a JavaScript project integrated with Google Sheets via Apps Script, providing a user-friendly interface for authoring and simulation of CF. The CF specification not only standardizes and modularizes molecular biology operations but also promotes collaboration, automation, and reuse, significantly reducing potential errors. The potential integration of CF with artificial intelligence, particularly GPT-4, suggests innovative automation strategies for synthetic biology. While challenges such as token limits, data storage, and biosecurity remain, proposed solutions promise a way forward in harnessing AI for experiment design. This shift from human-driven design to AI-assisted workflows, steered by high-level objectives, charts a potential future path in synthetic biology, envisioning an environment where complexities are managed more effectively.

59 BASIC BIOLOGICAL SCIENCES↗

Plant synthetic biology as a tool to help eliminate hidden hunger

Agricultural systems are under increasing pressure from declining environmental conditions, a growing population, and changes in consumer preferences, resulting in widespread malnutrition-related illnesses. Improving plant nutritional content through biotechnology techniques such as synthetic biology is a promising strategy to help combat hidden hunger caused by the lack of affordable and healthy foods in human diets. Production of compounds usually found in animal-rich diets, such as vitamin D or omega-3 fatty acids, has been recently demonstrated in planta. Here, we review recent biotechnological approaches to biofortifying plants with vitamins, minerals, and other metabolites, and summarise synthetic biology advances that offer the opportunity to build on these early biofortification efforts.

59 BASIC BIOLOGICAL SCIENCES↗

Synthetic Biology of Plants and Microbes for Agriculture, Environment, and Future Applications

Agriculture is under pressure to provide food for a growing population and the feedstock required to drive the bioeconomy. Methods to breed and genetically modify plants are inadequate to keep pace. When engineering crops, traits are painstakingly introduced into plants one-at-a-time, combine unpredictably, and are continuously expressed. Synthetic biology is changing these paradigms with new genome construction tools, computer aided design (CAD), and artificial intelligence (AI). “Smart plants” contain circuits that respond to environmental change, alter morphology, or respond to threats. Further, the plant and associated microbes (fungi, bacteria, archaea) are now being viewed by genetic engineers as a holistic system. Historically, plant health has been enhanced by many natural and laboratory-evolved soil microbes marketed to enhance growth, provide nutrients, or confer pest/stress resistance. Synthetic biology has expanded the number of species that can be engineered, increased the complexity of engineered functions, controlled environmental release, and assembled stable consortia. New CAD tools will manage genetic engineering projects spanning multiple plant genomes (nucleus, chloroplast, mitochondrion) and the thousands of genomes of associated bacteria/fungi. Here, this review covers advanced genetic engineering techniques to drive the next agricultural revolution, as well as push plant engineering into new realms for manufacturing, infrastructure, sensing, and remediation.

Clauer, Phillip [Massachusetts Inst. of Technology↗

Agrobacterium tumefaciens : A Bacterium Primed for Synthetic Biology

Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation.

59 BASIC BIOLOGICAL SCIENCES↗

Plant Synthetic Biology Conference – Travel Awardees and Speakers

The overarching goals of the Plant Synthetic Biology (SynBio) Conference, convened by the American Society of Plant Biologists (ASPB), were to raise community consciousness of what SynBio is and what it can do in key areas, including crop productivity, specialty chemicals, biosensors, and microbe-plant interactions. Within that context, the conference had two broad objectives. First, to feature a set of leading university and industry speakers from the US and Europe whose research on plants and microbes spans the SynBio spectrum with respect both to technology and to applications, as well as short talks (selected from submitted abstracts) on cutting-edge topics. And second, to foster exchanges among public sector scientists and industry scientists, whose participation as poster presenters, exhibitors, and sponsors was actively encouraged because so much innovative SynBio work is being carried out at dynamic young companies.

60 APPLIED LIFE SCIENCES↗

An argument for using anaerobes as microbial cell factories to advance synthetic biology and biomanufacturing

Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value-added bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

Plant synthetic biology innovations for biofuels and bioproducts

Plant-based biosynthesis of fuels, chemicals, and materials promotes environmental sustainability, which includes decreases in greenhouse gas emissions, water pollution, and loss of biodiversity. Advances in plant synthetic biology (synbio) should improve precision and efficacy of genetic engineering for sustainability. Applicable synbio innovations include genome editing, gene circuit design, synthetic promoter development, gene stacking technologies, and the design of environmental sensors. Moreover, recent advancements in developing spatially resolved and single-cell omics contribute to the discovery and characterization of cell-type-specific mechanisms and spatiotemporal gene regulations in distinct plant tissues for the expression of cell- and tissue-specific genes, resulting in improved bioproduction. In conclusion, this review highlights recent plant synbio progress and new single-cell molecular profiling towards sustainable biofuel and biomaterial production.

Yang, Yongil↗

Leveraging a synthetic biology approach to enhance BCG-mediated expansion of Vγ9Vδ2 T cells

There is an urgent need to develop a more efficacious anti-tuberculosis vaccine as the current live-attenuated vaccine strain BCG fails to prevent pulmonary infection in adults. In this study, we leverage a synthetic biology approach to engineer BCG to produce more (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate of bacterial—but not host—isoprenoid biosynthesis via the methylerythritol phosphate (MEP) pathway. HMBPP strongly activates and expands Vγ9Vδ2 T cells, which are unique to higher-order primates and protect against Mycobacterium tuberculosis infection. BCG has been engineered to produce specific ligands and antigens to some success; in contrast, our strategy exploits a self-nonself recognition mechanism in the host via HMBPP sensing, which has not been attempted before. To inform the design of our recombinant strains, we performed synteny analyses of >63 mycobacterial species and found that isoprenoid biosynthetic genes are not operonic across all the 356 surveyed genomes, but some genes are frequently found in pairs. Thus, we generated synthetic loci with the goal of specifically overproducing HMBPP and tested the ability of these engineered strains to induce human Vγ9Vδ2 expansion in an in vitro stimulation assay. We found that BCG expressing a synthetic MEP locus significantly enhanced Vγ9Vδ2 T cell expansion over the wild-type vaccine strain, and overexpression of the HMBPP synthase GcpE alone potently induced Vγ9Vδ2 T cell expansion with no downregulation of other pathway genes. Together these engineered strains present two successful strategies to accumulate HMBPP and overcome feedback inhibition of the MEP pathway.

59 BASIC BIOLOGICAL SCIENCES↗

Wolffia, a minimalist plant and synthetic biology chassis

A highly simplified species for genome engineering would facilitate rational design of a synthetic plant. A candidate species is the aquatic, non-grass monocot wolffia (Wolffia australiana) in the Lemnaceae family. Commonly known as watermeal, wolffia is a rootless ball of several thousand cells the size of a pinhead and the fastest growing plant known on Earth. Its extreme morphological reduction is coupled to transposon-mediated streamlining of its transcriptome, which represents a core set of nonredundant protein coding genes. Despite its body plan and transcriptome being highly specialized for continuous growth, wolffia retains cell types relevant to higher plants. Finally, systems level studies with this species could enable the creation of a defined biological chassis for synthetic plant construction.

59 BASIC BIOLOGICAL SCIENCES↗

Hijacking Translation Initiation for Synthetic Biology

Genetic code expansion (GCE) has revolutionized the field of protein chemistry. Over the past several decades more than 150 different noncanonical amino acids (ncAAs) have been co-translationally installed into proteins within various host organisms. Further, the vast majority of these ncAAs have been incorporated between the start and stop codons within an open reading frame. This requires that the ncAA be able to form a peptide bond at the α-amine, limiting the types of molecules that can be genetically encoded. In contrast, the α-amine of the initiating amino acid is not required for peptide bond formation. Therefore, including the initiator position in GCE allows for co-translational insertion of more diverse molecules that are modified, or completely lacking an α-amine. This review explores various methods which have been used to initiate protein synthesis with diverse molecules both in vitro and in vivo.

59 BASIC BIOLOGICAL SCIENCES↗