5

Cases Across the Continuum: Biosafety, Biosecurity, and Environmental Considerations

Building on the conceptual framework and definitions established in Chapter 2, the analytical approaches for evaluating risk, benefit, and uncertainty outlined in Chapter 3, and the governance landscape and potential gaps described in Chapter 4, this chapter applies those concepts to a set of illustrative cases spanning the synthetic cell continuum (Table 5-1). These cases range from simple, non-replicating biochemical assemblies to genome-containing systems intended for environmental or societal use, and they are designed to reflect increasing complexity, functionality, and potential exposure pathways.

The cases were selected to capture key inflection points along the synthetic cell continuum where governance questions meaningfully shift, rather than to provide an exhaustive survey of possible applications. Together, they demonstrate how biosafety, biosecurity, and environmental considerations change across this continuum, and how design choices and context of use shape governance needs. In particular, they illustrate how the core evaluative questions introduced earlier in the report—whether a synthetic cell contains genetic material, whether it can replicate, how and where it is intended to be used or deployed, and whether it raises biosecurity concerns (see Box 2-1)—trans-late into practical decisions about containment, oversight, regulatory jurisdiction, and communication.

These cases provide a comparative lens for examining governance readiness, identifying recurring oversight gaps, and highlighting principles for proportionate, adaptive management of synthetic cell research and applications. By juxtaposing cases at different points along the continuum (see Figure 2-1), the chapter clarifies where existing frameworks are generally sufficient, where interpretation varies across institutions or agencies, and where emerging synthetic cell designs may challenge current biosafety, biosecurity, or environmental governance approaches. Case-based analyses in other areas of emerging biotechnology have similarly demonstrated how applying

existing biosafety and biosecurity policies to novel technological capabilities can clarify oversight pathways and reveal gaps, including with respect to environmental and cross-domain considerations (DiEuliis et al., 2017; Evans and Palmer, 2018; Evans et al., 2020; NASEM, 2016, Chapter 3).

Consistent with the committee’s charge to identify potential gaps in existing biosafety, biosecurity, and environmental oversight of possible synthetic cell applications (see Box 1-1, Statement of Task), ethical, cultural, economic, and broader societal dimensions of the cases were not systematically evaluated. For this reason, the report does not systematically evaluate questions of whether synthetic cell approaches are the best or most appropriate solutions to existing problems, or whether they are likely to be supported or embraced by stakeholders, users, and communities. At the same time, the committee recognizes that questions of risk, benefit, and uncertainty are not purely technical. Risk assessments integrate both technical knowledge and values-based priorities, and effective governance benefits from making explicit the sources of knowledge, underlying assumptions, and the processes by which priorities are established. Where relevant, the chapter identifies considerations related to transparency, stakeholder engagement, and public trust; these considerations are discussed further in Chapter 6 in the context of responsible innovation.

CASE 1 – BASIC RESEARCH: CHEMOTAXIS

In this real-world example from recent literature, researchers built a simple cell-like system capable of chemotaxis, defined here as directed movement toward a specific chemical stimulus (Borges-Fernandes et al., 2025). The system consists of a lipid bilayer vesicle encapsulating a single enzyme (either glucose oxidase or urease) and incorporating the transmembrane pore protein, α-hemolysin, a commonly used component in synthetic cell engineering. The construct contains no genetic material and cannot replicate.

Despite this simplicity, the system demonstrates a cell-like behavior—directed movement toward certain chemicals (glucose or urea)—highlighting how biological functions can emerge from minimal biochemical assemblies. By stripping cellular function to its most basic elements, such constructs enable researchers to test hypotheses about the origins of life and study how cellular behaviors emerge from molecular interactions.

Within the synthetic cell continuum, this case represents an extremely simple, non-genome containing design and sits at the lowest end of the technology-readiness spectrum (Technology Readiness Level [TRL] 1-2; see Table 4-1). Its primary purpose is fundamental research rather than application development, making it a useful boundary case for examining how existing oversight frameworks apply to life-like but non-living systems.

TABLE 5-1 Synthetic Cell Cases Across the Continuum

Risk Considerations

Biosafety

Systems that lack genetic material yet exhibit life-like behavior may fall ambiguously between chemical and biological oversight. Because this construct contains no genetic material and cannot replicate, it may fall outside of some institutions’ biosafety categorization schemes, creating variability in terms of whether and how it is reviewed. Some institutions may not classify this system as needing biosafety oversight. Depending on the institution, the synthetic cell may not be assigned a risk group

(RG) or biological safety level (BSL) classification, or may not be reviewed through biosafety mechanisms at all (see Box 4-1, Tables 4-2 and 4-3). From a hazard perspective, the chemotactic vesicle has no pathogenic potential and cannot reproduce, and thus most closely resembles laboratory systems involving basic chemicals rather than biological agents. At some institutions, it may therefore be treated solely as a chemical system rather than as a biological one, particularly because it contains no genetic elements, which reduces concern about horizontal gene transfer or unanticipated metabolic activity. However, because uncertainty remains regarding how novel constructs might interact with natural systems or laboratory environments, some institutional biosafety committees (IBCs), which review and oversee research involving recombinant or synthetic nucleic acids and other biological hazards, might still consider BSL-2 containment and procedures appropriate in the early stages of research, particularly to account for administrative controls and human factors in handling novel systems.

This range in interpretations illustrates a potential governance gap: As systems shift from purely chemical constructs to entities with cell-like behaviors, they may not map cleanly onto existing risk group and biosafety level categories, leading to inconsistent oversight outcomes across institutions. If recombinant or synthetic nucleic acids are used to produce system components (for example, expression of α-hemolysin), those upstream steps may trigger IBC review and National Institutes of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines; NIH, 2024) applicability even though the final construct contains no nucleic acids. Some institutions require the use of α-hemolysin, a potent protein toxin, to be conducted in BSL-2 containment, while other institutions may allow its use at low concentrations in a basic chemical laboratory or a BSL-1 facility.

Chemical Safety

Since one component of this synthetic cell, α-hemolysin, is a biologically derived toxin, this component would be assessed under institutional hazardous chemical and toxin regulations, which may require additional oversight or handling restrictions. Although most reagents in the system are low hazard, proper evaluation of potential irritants, reactive byproducts, and toxin-related exposure pathways is essential. Standard chemical hygiene practices, combined with any toxin-specific institutional requirements, are generally sufficient to manage risk.

Biosecurity

This system poses negligible dual use or misuse potential and therefore is of minimal biosecurity concern. The primary biosecurity relevance is indirect and capability-based: The methods and know-how contribute to the broader synthetic cell toolkit, even if this specific construct is not particularly suitable for misuse.

Ecological Considerations

These synthetic cells are expected to degrade and not persist, although possible interactions with microbial communities or persistence of lipid or enzyme components warrant some consideration. No genetic material is present for possible horizontal gene transfer into environmental organisms. Because the work is intended for contained laboratory observation rather than environmental release, the main ecological considerations relate to accidental release, persistence of materials, and decontamination and disposal practices.

Governance Considerations

This case challenges existing oversight frameworks because the synthetic cell lacks DNA yet displays a life-like behavior, raising questions about whether chemical or biological safety oversight should apply and how such constructs should be categorized in research policy. Although the construct itself does not contain recombinant or synthetic nucleic acids, some steps (such as producing α-hemolysin via recombinant expression) may fall under IBC review and, at covered institutions, relevant provisions of the NIH Guidelines. Conceptually, this case highlights distinctions between biological systems and chemistry, raising questions about how simple, life-like systems should be defined and overseen in governance frameworks.

The minimal chemotactic vesicle serves as a test case for understanding how current oversight frameworks apply to synthetic cells. While the direct risks to human health, security, and the environment appear negligible, the case raises broader questions: Does a non-replicating system with cell-like behavior require governance beyond chemical safety? Should oversight focus solely on immediate hazards, or also on the incremental technical capabilities that the research confers? In addition, how should such systems be categorized and communicated, given that their simplicity challenges conventional definitions of what constitutes a “cell”? As a boundary case on the synthetic cell continuum, this example underscores the value of function- and context-based criteria (rather than DNA presence alone) for determining when biosafety, biosecurity, or environmental review is warranted. This case uniquely spotlights non-genetic systems that are exempt under current recombinant DNA–based oversight yet still raise questions about how far biosafety and environmental governance may extend into the chemical domain.

CASE 2 – CONSUMER PRODUCT: HOUSEHOLD HAZARD DETECTION

In this fictitious example, a biotechnology start-up has developed a synthetic cell designed for consumer use to detect hazardous mold spores in household environments. The user sprays a solution containing these synthetic cells onto a surface, and a visible color change indicates the presence of mold that may not be detectable by eye.

The synthetic cells contain a minimal set of engineered genes that enable detection of multiple types of mold and production of a colorimetric signal. They do not contain a complete genome and are not capable of growth or replication. The synthetic cells are designed to be shelf-stable, tunable for specific detection targets, and incapable of evolution.

This case represents a mid-range example on the synthetic cell continuum: It incorporates genetic material and programmed biological function, yet is intentionally non-replicating and designed for use outside of laboratory or clinical settings. Unlike Case 1, which is confined to basic research, this example introduces direct interaction with public environments, expanding the range of exposure pathways and governance considerations.

This hypothetical example of synthetic cell technology presents a new approach to hazard detection, reduced risks compared to live organisms, and scalable platforms that can be extended to other kinds of applications. From a technology-readiness perspective, this is a mid-range example (approximately TRL 4-7; see Table 4-1), reflecting the transition from laboratory development toward a deployable consumer product. As such, the case illustrates how synthetic cells may blur boundaries between chemical consumer products and quasi-biological systems, and how relatively small design choices, such as the encapsulation of enzymes versus genetic material, can substantially affect oversight pathways.

Risk Considerations

Biosafety

These constructs may not easily fit into existing risk group categories. Although the synthetic cells are designed to be non-replicating and lack a complete genome, they do contain engineered genetic material and express biologically active products, placing them closer to the biological end of the synthetic cell continuum than purely chemical systems. Uncertainties remain regarding how they interact with living systems within their intended deployment environment, such as humans or household pets, given their intended consumer use outside traditional laboratory settings. IBCs help to ensure appropriate containment practices and compliance with applicable biosafety requirements (including, where relevant, the NIH Guidelines). IBCs oversee early research to carefully weigh laboratory practices, with some applications potentially warranting BSL-2 containment and precautions despite a low apparent risk, as indicated by a standard risk assessment that considers (among other factors): whether any component can replicate or persist; whether expressed products include toxins or biologically active effectors; likely routes of exposure during use (aerosol generation from spraying, skin or eye contact, ingestion); expected dose and frequency; environmental fate (degradation versus persistence); and the availability of effective decontamination and spill response measures. In practice, uncertainty related to potential aerosol exposure and unintended biological activity can justify BSL-2 work practices, even when the construct is designed to be non-replicating and low hazard. Where the product relies on encapsulated transcription-translation systems and engineered DNA, the associated research at covered institutions would generally fall within the NIH Guidelines, although some versions of the systems might qualify for partial exemptions if their nucleic acids cannot replicate in any living cell. By contrast, otherwise similar enzyme-only designs would fall outside the NIH Guidelines, highlighting how subtle molecular design choices can place functionally similar consumer products on very different oversight pathways.

Chemical Safety

These formulations may include small-molecule dyes, solvents, stabilizers, surfactants, and other conventional ingredients. Accordingly, chemical safety considerations such as irritation or sensitization, inhalation hazards from spraying, chronic low-level exposure, and disposal pathways would be evaluated under standard consumer-chemical and occupational safety frameworks, alongside any biotechnology-specific oversight.

Biosecurity

In this specific mold-detection application, the synthetic cell is engineered to produce only a visual color change and is not designed to generate or release biologically active substances. The commercial product itself therefore presents little credible biosecurity risk. Any biosecurity relevance arises indirectly, through the broader platform capabilities and programmable molecular components (such as encapsulated transcription-translation systems and engineered nucleic acids). Repurposing such systems for harmful outputs would require substantial redesign, specialized expertise, and controlled manufacturing capabilities well beyond what is accessible through consumer products. As a result, any biosecurity considerations in this case are best understood as forward-looking and capabilities-based, rather than a realistic misuse pathway associated with the mold-detection product itself.

Ecological Considerations

Synthetic cells used on household surfaces may be washed into wastewater systems or disposed as municipal solid waste. While the cells in this hypothetical example are designed to degrade, uncertainties remain about their persistence, stability, and ecological effects, especially in the absence of baseline environmental data. Even non-replicating constructs may interact transiently with microbial communities or contribute genetic material to environmental matrices, highlighting the relevance of downstream fate and transport considerations despite the absence of intentional environmental release.

Governance Considerations

Responsibility for regulatory oversight in this example would be distributed across multiple agencies under existing statutory authorities (OSTP, 1986, 2017). For a consumer household spray containing engineered genetic material, the Environmental Protection Agency (EPA) would likely have authority under the Toxic Substances Control Act (TSCA)¹ if the construct qualifies as a “new chemical substance” or falls within EPA’s biotechnology regulations governing intergeneric microorganisms (EPA, 2012). EPA has regulated sensors under TSCA, including those that incorporate recombinant DNA or other intergeneric genetic elements. If the product makes pesticidal claims, such

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¹ 15 U.S.C. § 2601 et seq. (1976).

as preventing, destroying, or mitigating mold, it would fall under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA),² which regulates chemical, biochemical, microbial, or related pesticidal products. The Consumer Product Safety Commission could also have jurisdiction over general consumer product safety, labeling, and injury hazards associated with household use. Occupational Safety and Health Administration (OSHA) requirements would govern worker exposure during manufacturing and formulation.

This case illustrates how synthetic cells designed for consumer use are regulated through established statutory frameworks that apply based on product composition, intended function, and claims, not on whether the product is described as “chemical” or “biotechnological,” but on how it meets statutory definitions and regulatory triggers. Under current law, these factors determine whether the product is reviewed under TSCA, FIFRA, or other applicable consumer and environmental statutes. Products with similar user experiences may therefore fall under different statutory authorities, underscoring the importance of early engagement with regulators to clarify jurisdiction and data requirements.

Public trust will depend not only on compliance with existing regulatory oversight, but also on transparent risk communication and clear expectations of how the constructs function and how they differ from living organisms or existing products on the market.

CASE 3 – BIOREMEDIATION: ENVIRONMENTAL CONTAMINANTS REMOVAL

This hypothetical case anticipates the development of synthetic cells designed for intentional environmental use, specifically to remove hazardous chemicals, such as phenols, from contaminated soil. These synthetic cells would operate by sensing target chemicals in their surroundings, importing those compounds across the cell boundary, and converting them into less harmful substances.

The design envisioned here relies on non-replicating and non-evolving synthetic cells, intended to provide a more controllable alternative to engineered living microorganisms for environmental remediation. The system is designed to reduce risks associated with genetic instability since it is devoid of functions such as division, mutation, and evolution. In this respect, the case illustrates how synthetic cells may be positioned as substitutes for engineered microbes in applications that traditionally require environmental release.

Orthogonal design techniques, such as the use of xenonucleic acids (XNAs) or nonstandard amino acids (NSAAs), could further limit unintended interactions with native organisms and reduce the likelihood of gene transfer. Their inherently limited viability may also promote more rapid environmental clearance following deployment. The simplified and modular nature of synthetic cells could enable predictive in silico modeling of performance and fate, potentially reducing the reliance on higher-risk field experimentation during development. Built-in fail-safes, such as engineered

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² 7 U.S.C. § 136-136y (1996).

self-termination mechanisms, could further enhance biosafety, making these synthetic cells a powerful tool for advancing biotechnology with reduced risk.

Within the synthetic cell continuum, this case occupies a transitional position: The system contains genetic material and performs complex biological functions, yet is intentionally non-replicating and designed for transient activity in open environments. Compared with Case 2, which involves consumer use with incidental environmental exposure, this example involves deliberate environmental deployment, elevating ecological considerations even in the absence of replication.

Other applications that could be envisioned for similar non-replicating synthetic cells include “smart” biosensors, targeted delivery of therapeutics for human or animal health, and programmable biocatalysts for chemical manufacturing, all of which could operate under controlled, time-limited conditions. However, even for non-replicating designs, packaged DNA components (for example, antibiotic resistance markers used during cloning) may remain susceptible to uptake by natural organisms through horizontal gene transfer, warranting consideration in environmental risk evaluations akin to considerations around DNA released from engineered cells.

Risk Considerations

Biosafety

Although they are non-replicating, the synthetic cells in this case contain DNA and express functional proteins; natural organisms could take up antibiotic resistance markers or other genetic parts via horizontal gene transfer. As a result, the absence of replication does not eliminate biosafety concerns related to genetic material, particularly when synthetic cells are deployed in open environments where exposure to natural microbial communities is expected. Degradation and persistence of DNA fragments in soil or water require evaluation. Contained research with encapsulated DNA constructs that retain the ability to replicate in standard laboratory hosts would typically fall within scope of the NIH Guidelines (often under Section III-E for RG1 work). Designs using nucleic acids that cannot replicate in any natural organism might instead qualify for exemption, illustrating how subtle design choices can shift nearly identical technologies into or out of the purview of the NIH Guidelines, which do not cover environmental releases. Instead, a permit may be required by the USDA Animal and Plant Health Inspection Service for field release or interstate transport. This distinction underscores a recurring governance challenge for synthetic cells: Oversight and regulatory triggers are often tied to genetic properties rather than to functional activity or exposure context.

Biosecurity

This case represents minimal direct dual use or misuse risk, given the non-replicating design and narrow, application-specific function. However, increasing sophistication in orthogonal genetic design and environmental deployment contributes incrementally to synthetic biology capabilities. As such, biosecurity relevance in this case is best

understood as capability-based and cumulative, rather than as arising from a credible misuse pathway associated with this specific application. This also highlights a limitation of case-by-case assessment: Individual projects may appear low risk while still advancing an innovation trajectory with clearer harmful or military applications. Accordingly, periodic review of emerging capabilities and their potential for misapplication is warranted even when a specific experiment does not itself raise immediate biosecurity concerns.

Chemical Safety

In this case, chemical safety extends beyond laboratory handling and worker exposure to include hazards arising from chemical transformations and material interactions following environmental deployment. Although the goal is detoxification, chemical risk does not disappear once target contaminants are taken up by synthetic cells. Incomplete or off-pathway conversion can generate hazardous intermediates or byproducts that retain toxicity or are more mobile in soil and water, and uptake may temporarily shift sorption or redox equilibria, increasing dissolved concentrations if degradation does not keep pace. Chemical safety considerations also include the substances required for system function. Membrane components, encapsulants, cofactors, or added reagents may introduce their own hazards or interact with local chemistry in unintended ways, such as altering metal mobility or generating reactive chemical species. As with conventional remediation technologies, assessment should focus on chemical fate, transformation products, and exposure pathways rather than assuming risk reduction solely from removal of the target contaminant.

Ecological Considerations

Possible unintended interactions with native microbiota or biogeochemical cycles warrant careful consideration, even for non-replicating systems. Intentional environmental deployment introduces exposure pathways that are absent in laboratory or consumer-use cases, elevating the importance of persistence, dispersal, and functional interaction with ecosystems. Even temporary activity may alter local microbial communities or environmental processes. Self-termination or “kill-switch” designs should be validated for robustness under realistic environmental variability including changes in temperature, moisture, nutrient availability, and microbial diversity. This case highlights a key limitation of existing biosafety frameworks: Ecological risks may arise from functional activity and material persistence rather than from pathogenicity or replication, suggesting the need for complementary ecological containment and monitoring approaches.

Governance Considerations

Environmental release of synthetic cells challenges existing regulatory boundaries between engineered organisms and chemical agents. Because the systems envisioned in

this case are intentionally non-replicating yet biologically active in open environments, they do not fit neatly within oversight frameworks that assume environmental risk is tied primarily to living, replicating organisms. As a result, governance may require hybrid oversight drawing on biotechnology, chemical safety, and environmental protection frameworks, rather than relying on a single, organism-centered regulatory pathway. In the face of such complexity and exposures of shared environments (e.g., soil, water, sediments), engagement of stakeholders and affected publics becomes an important component of governance (NASEM, 2016, Chapter 9).

Contained research involving recombinant or synthetic nucleic acids would be covered under the NIH Guidelines and reviewed by IBCs. However, once the focus shifts from laboratory development to intentional environmental deployment, the relevance of the NIH Guidelines diminishes, and questions of federal regulatory jurisdiction become more prominent. Regulators may need to determine whether such constructs should be regulated as transgenic microorganisms or as new chemical substances under EPA’s TSCA authority. This illustrates a potential gap in current regulations that may require clearer guidance on environmental assessment, post-deployment monitoring, and criteria for ecological containment for non-living yet biologically functional systems.

Although the synthetic cells in this case are not living organisms, they are designed to carry out active biochemical functions in soil and water, potentially interacting with native microbial communities and environmental processes. Assigning responsibility for oversight, monitoring, and remediation therefore depends less on whether the system is alive and more on its functional activity, persistence, and exposure pathways.

As a mid-continuum case, this example highlights a gap in existing governance frameworks: There is no clearly defined mechanism for evaluating and managing ecological risks posed by non-living yet biologically functional systems intended for environmental use. Addressing this gap may require clearer guidance on environmental assessment, post-deployment monitoring, and criteria for ecological containment that operate alongside, rather than within, traditional biosafety level classifications.

CASE 4 – MEDICINE: TARGETED GASTROINTESTINAL THERAPY

In this fictitious case, a medical university is developing a synthetic cell for therapeutic use in humans, specifically the treatment of infections in the digestive tract. The synthetic cells are packaged in dehydrated form within capsules, swallowed by the patient, and rehydrated during digestion, activating the encapsulated system.

Upon rehydration, the synthetic cells initiate transcription and translation using a limited set of DNA instructions, rather than a full genome. The system is intentionally non-replicating and designed to function transiently within the gastrointestinal environment. The synthetic cells sense the presence of a specific pathogenic bacterium, and only when that pathogen is detected do they produce an antibiotic intended to combat the infection.

This targeted, conditional activity represents a key design rationale for synthetic cell therapeutics. Compared with conventional orally administered antibiotics, which are broadly distributed throughout the gut, this approach aims to localize antibiotic

production to the site of infection, thereby reducing overall antibiotic exposure and helping preserve beneficial components of the patient’s gut microbiota.

Within the synthetic cell continuum, this system is mid-range, incorporating genetic material and biologically active functions, yet remains non-replicating and is designed for controlled, internal use within the human body. The case introduces direct, intentional human exposure, distinguishing it from Cases 1–3 and elevating considerations related to clinical safety, regulatory oversight, and downstream environmental pathways.

From a technology readiness perspective, this is a translational example (approximately TRLs 4–7; see Table 4-1), reflecting preclinical development and early testing rather than approved clinical use. As such, the case illustrates how synthetic cells may enter regulated medical product pathways while still raising questions distinct from those posed by conventional biologics or small-molecule drugs.

Risk Considerations

Biosafety

As these cells are non-replicating, they are not expected to be capable of infection or long-term colonization. However, because they are designed to function within the human body and to produce antibiotic molecules, they introduce biosafety considerations distinct from those associated with contained laboratory research. Accidental exposure during manufacturing, handling, or administration could result in unintended biological effects, particularly if dosage, activation conditions, or degradation pathways are not fully characterized. Because the capsules contain engineered DNA and transcription machinery, most early-stage research at institutions covered by the NIH Guidelines would fall within the scope of those guidelines. Risk assessment would require consideration not only of pathogenicity, but also expression products, exposure routes, and failure modes within the gastrointestinal environment.

Biosecurity

The potential for misuse of these specific synthetic cells with their intended application is expected to be low. Nevertheless, the platform’s ability to sense biological signals and produce active compounds raises broader biosecurity considerations, such as the prospect that similar cells could be developed to enable the delivery of toxins or other biomolecular cargo. The misuse potential in this case depends more on what is produced and delivered than on the non-replicating synthetic cells themselves. As such, biosecurity relevance is best understood as capability-based and forward-looking, rather than tied to a plausible misuse pathway associated with the therapeutic application as designed.

Ecological Considerations

Although designed for use within the human body, components of these synthetic cells, including residual DNA, proteins, and lipid materials, would likely enter wastewater systems after excretion. As with other engineered biological systems used in medicine and with discarded pharmaceutical products, this introduces secondary environmental consideration, including the fate and persistence of genetic material, the potential for horizontal gene transfer to environmental microorganisms, and the possible uptake or ingestion of synthetic cell components or byproducts by animals, plants, or other microbes. While replication is not expected, downstream environmental exposure pathways remain relevant and warrant consideration in life cycle–based risk assessments.

Governance Considerations

The Food and Drug Administration (FDA) would likely be the lead regulator for a therapeutic product of this type, since it functions as a treatment delivered to patients. Depending on its classification, the product could be regulated as a drug, biologic, combination product, or novel therapeutic modality, with oversight focused on safety, efficacy, manufacturing quality, and clinical performance.

Contained research that involves recombinant or synthetic nucleic acids would remain under the NIH Guidelines and would be reviewed by local IBCs. As the system progresses toward clinical testing and manufacturing, oversight would increasingly shift from institutional biosafety mechanisms to FDA-regulated pathways, which would include Good Manufacturing Practice standards and requirements. Workplace safety during production would fall under OSHA requirements.

This case illustrates how synthetic cells intended for medical use can move across governance regimes as they progress from research to application, transitioning from biosafety-focused oversight toward product-based regulatory review. At the same time, the hybrid nature of the construct—non-living yet biologically functional—may complicate classification under existing regulatory categories, particularly if the system does not align cleanly with traditional definitions of drugs, biologics, or devices. This scenario shows how future therapeutics based on synthetic cells may require continued coordination across drug, biologic, workplace safety, and environmental safety systems, even when the underlying constructs cannot replicate.

As a mid-continuum case involving direct human exposure but no replication, this example underscores the importance of life cycle–based governance. This case also reinforces that governance frameworks must integrate biosafety, biosecurity, environmental, and product-regulatory considerations to support proportionate and adaptive oversight as synthetic cells move toward real-world use.

CASE 5 – AGRICULTURE: CROP NUTRIENT DELIVERY

This hypothetical case envisions a research consortium developing a replicating synthetic cell intended for agricultural use, specifically to improve crop yields and

reduce fertilizer use by enhancing nutrient availability in soil. Unlike conventional fertilizers, which are applied broadly and often inefficiently, this synthetic cell is engineered to sense chemical signals from the roots of specific crop plants and release nutrients in precise, localized amounts in response. Other nitrogen-fixing products on the U.S. market use naturally occurring or genetically engineered microorganisms rather than synthetic cells (Kula Bio, n.d.; Pivot Bio, n.d.).

The synthetic cells envisioned here are designed to migrate through soil toward target plant roots and to replicate only in the presence of those roots, creating an engineered symbiosis that limits replication to a defined ecological location. Early greenhouse trials demonstrating promising outcomes, including reduced fertilizer requirements, decreased fertilizer runoff into water systems, and improved crop performance under stress conditions, would show how such a system could advance goals of sustainable agriculture and climate mitigation while reducing costs for farmers. Later applications of this platform could include customization to produce distinct nutrient profiles for different crops or the production of nutrient supplements for livestock.

This case represents a transition to higher biological complexity and autonomy. Unlike Cases 1–4, which involve non-replicating systems, this example incorporates controlled replication and long-term interaction with living organisms and ecosystems. As such, it introduces a qualitatively different set of biosafety, biosecurity, and ecological considerations.

From a technology readiness perspective, this is a mid-stage example because it involves controlled replication and early greenhouse tests, which introduce questions about how such systems might behave in more complex outdoor environments. The case illustrates the governance challenges that emerge as synthetic cells move from contained research toward applications involving sustained environmental presence and ecological interaction.

Risk Considerations

Biosafety

Under the current NIH framework, laboratory work to engineer and test the synthetic cells would likely fall under RG1, since the synthetic cells are not expected to pose direct pathogenic risk to humans. Contained research may include activities in laboratories or in greenhouses with Biological Safety Level 1 for plants (see Appendix L-II of the NIH Guidelines). However, the ability of the system to replicate, albeit under defined conditions, introduces additional uncertainty regarding stability, behavior, and the potential for unintended interactions. IBCs may therefore face interpretive challenges in assigning risk categories and determining appropriate containment practices for early-stage research. For institutions covered by the NIH Guidelines, this work would typically fall under the sections governing plant research involving recombinant DNA. As development progresses beyond the laboratory and greenhouse toward outdoor testing, traditional biosafety classifications may become less informative and other factors, such as risks to wildlife, may become relevant. The specific formulation

and classification of the product (e.g., as a soil amendment, fertilizer, or other product category) will have implications for which agencies are involved in regulatory oversight, marketing, and release permits.

Biosecurity

Although the intended application is benign and agriculturally beneficial, the platform could be misused if adapted to produce harmful metabolites or if engineered for persistence in unintended hosts. The dual use potential of programmable synthetic cells, even in agricultural settings, raises questions about whether existing oversight is sufficient and how private-sector developers are engaged in responsible stewardship. Although these risks are not unique to synthetic cells, they become more prominent when designs allow replication outside the laboratory.

Ecological Considerations

The greatest uncertainties in this case relate to potential environmental interactions and their effects. Replication in soil environments raises questions about persistence, spread beyond unintended zones, and interactions with native microbial communities. Potential impacts include alterations to soil microbiomes, effects on nutrient cycling and biogeochemical processes, and unintended interactions with non-target organisms such as beneficial soil microbes, invertebrates, or pollinators. What happens if the synthetic cells persist longer than intended, evolve to replicate without having to be in the presence of crop roots, or encounter environmental conditions that expand their viable habitat? Could they migrate into waterways or adjacent ecosystems through runoff or soil transport? These uncertainties highlight the potential utility of ecological containment strategies that operate alongside genetic safeguards, including monitoring requirements, spatial and temporal limits on deployment, and mechanisms for recovery or neutralization if spread occurs beyond intended boundaries.

Governance Considerations

In the United States, oversight of such a product could be distributed across multiple agencies under the Coordinated Framework. Depending on the product classification, EPA might conduct environmental release and ecological risk assessment under statutes such as TSCA. USDA might be involved in evaluating plant health and agricultural impacts. If residues entered the food chain, FDA authorities would also become relevant. EPA and USDA may require approvals for greenhouse studies and field trials, including the review of data on environmental fate, persistence, and ecological interaction. Such distributed oversight underscores the importance of interagency coordination, clarity for developers, and the engagement of stakeholders and relevant publics (e.g., farmers, consumers). This challenge is not unique to synthetic cells but extends to engineered microbes for environmental release and related biotechnology products.

As a higher-complexity case on the synthetic cell continuum, this example highlights a gap in existing governance frameworks: While biosafety systems are well

developed for contained laboratory research, no integrated approach exists for assessing and managing ecological risks associated with replicating synthetic systems designed for open environments. Ecological containment guidance, including criteria for genetic safeguards, phased testing, long-term monitoring, and post-deployment stewardship, may help to address this gap. More broadly, this case underscores the need for oversight approaches that extend beyond narrow risk group classifications and the importance of integrating biosafety, biosecurity, and ecological considerations.

CASE 6 – BIOMANUFACTURING: ROBUST PROTEIN MANUFACTURING

This fictitious case envisions the development of a bacterial strain optimized for industrial biomanufacturing, based on a non-pathogenic K-12 strain of Escherichia coli but with a fully synthetic, extensively refactored genome. The organism is engineered to retain many core biological features of its natural counterpart, including its membrane structure, metabolism, and capacity for replication, while introducing a genetic code different from any found in nature.

This case is rooted in multiple proof-of-principle studies in genome engineering (Lajoie et al., 2013; Pósfai et al., 2006) and synthetic genomics demonstrations (Fredens et al., 2019; Hutchinson et al., 2016). Reducing a bacterial genome to remove unstable genetic elements has the potential to improve the reliability of biomanufacturing processes (Umenhoffer et al., 2010). Rewiring the genetic code can impart resistance (Ma and Isaacs, 2016) or even immunity (Nyerges et al., 2023) to infection by bacteriophages that can cause the failure of production runs. Reduced genetic codes also provide the opportunity to incorporate NSAAs into proteins at high yield, expanding the functional range of protein design (Huang et al., 2025). Most recently, a novel E. coli strain, Syn57, was engineered with a fully synthesized genome (Robertson et al., 2025). In that case, researchers reduced the genome size by more than 10 percent, eliminated six redundant sense codons and one stop codon, and replaced those codons with synonymous alternatives. Some researchers would describe such organisms as synthetic cells, while others would categorize these examples separately as synthetic genomics. For the purposes of this report, the case is included to examine governance implications at the boundary between synthetic cells and highly engineered living organisms.

Within the synthetic cell continuum, this hypothetical case represents a high-complexity, genome-containing, replicating system intended for long-term operation under contained industrial conditions. Unlike Case 5, which involves environmental deployment, this example emphasizes containment, scale, and persistence within engineered facilities, highlighting a different set of biosafety, biosecurity, and governance considerations.

From a technology readiness perspective, this is a mid- to late-stage example, reflecting advanced laboratory development and pilot-scale manufacturing rather than unrestricted deployment. The case illustrates how synthetic cells may be used to improve industrial performance while simultaneously challenging assumptions embedded in existing oversight frameworks.

Risk Considerations

Biosafety

Although derived from a non-pathogenic, RG1 E. coli strain, the wholly synthetic genome and novel codon usage introduce uncertainty about this synthetic cell’s behavior under non-laboratory conditions. Extensive genome refactoring may alter gene expression, metabolic regulation, or stress responses in ways that are difficult to predict based on experience with conventional strains. Because it is a K-12 strain of E. coli, this bacterium may be exempt from the NIH Guidelines (see Appendix C-II of the NIH Guidelines). The biosafety level would be determined by an institution’s IBC depending on what is cloned into the E. coli. Some institutions may adopt more conservative containment practices given the degree of genome rewriting and novelty of the genetic code.

Biosecurity

The construct’s resistance to bacteriophage infection and potential for orthogonal genetic isolation reduce some conventional risks. At the same time, these same features could complicate detection, attribution, or control if the organism were misused or escaped containment. Knowledge and tools developed for large-scale genome refactoring may have dual use implications as the technology advances. These concerns extend beyond this single organism and relate to the growing capabilities of synthetic genomics as a whole.

Ecological Considerations

Although the synthetic cell in this example is intended for use in contained biomanufacturing facilities, accidental release into the environment cannot be fully excluded. A modified genetic code may affect interactions with natural microbial communities. The recoded genome might reduce effects of horizontal gene transfer, but there are uncertainties surrounding the possibility of environmental persistence of DNA or partial compatibility with other organisms. Immunity to phage infection could provide a special competitive advantage over other microbes in the environment.

Governance Considerations

Because this case is intended for industrial biomanufacturing, it would likely fall under the authority of EPA pursuant to TSCA, which regulates certain new chemical substances and intergeneric microorganisms used for commercial purposes.³ A genome-refactored E. coli strain containing synthetic genetic elements not found in nature may qualify as a “new microorganism” under TSCA biotechnology regulations,

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³ 40 CFR Part 725.

potentially requiring submission of a Microbial Commercial Activity Notice prior to commercial manufacture. EPA review would evaluate potential risks to human health and the environment associated with large-scale production, containment practices, worker exposure, waste management, and possible accidental release. Unlike the NIH Guidelines, which apply based on funding and institutional coverage, TSCA authority applies regardless of funding source and provides a statutory mechanism for premanufacture review and risk management conditions for industrial biotechnology applications such as this case.

Using a fully synthetic, refactored genome, this case could challenge current oversight systems, which essentially assume natural genetic architectures. Such extremely engineered organisms blur traditional definitions of “natural life,” prompting discussion of synthetic life boundaries, ownership, and stewardship responsibilities in the design of alternative genetic codes (see discussion in Chapter 2 on Clarifying Boundaries and Blurred Lines). This case raises broader questions about how to classify and evaluate organisms whose genomes have been partially or completely redesigned and what stewardship responsibilities accompany such capabilities.

CASE 7 – BASIC RESEARCH: ORIGINS-OF-LIFE SYNTHETIC CELL

A central goal of much synthetic cell research is to produce a fully living, self-replicating cell from non-living components (Rothschild et al., 2024), i.e., using a “bottom-up” approach. This hypothetical case anticipates the achievement of that goal. The principal application in mind would be fundamental knowledge—confirmation of the components that define a living system with implications for understanding the origins of life on this planet. This example envisions a synthetic cell created using the JCV-syn3.0 synthetic minimal genome (Hutchinson et al., 2016) that is able to replicate independently in culture media despite having only 473 genes encoded in 531 kilobase pairs of DNA.

This envisioned organism, derived from the bacterium Mycoplasma mycoides, essentially provides one version of a “parts list” for the genes needed to produce a simple living cell. Although no such reconstruction has yet occurred in the real world, this hypothetical synthetic cell may be attainable by the combination of: 1) DNA-free cell extracts derived from M. mycoides containing the molecular machinery needed for transcription and translation; 2) the synthetic DNA genome of JCV-syn3.0; 3) small-molecule building blocks, metabolites, and energy sources; and 4) a lipid bilayer of appropriate composition. These elements might successfully be combined using microfluidics to control the packaging of the first three components into cell-sized vesicles contained within a lipid bilayer. At this stage it is unknown whether such a combination could attain a living, replicating state as is, or whether an additional step (some “spark of life”) would be required.

This synthetic cell would be expected to have all cellular features possessed by the JCV-syn3.0-derived strain developed by Hutchinson, et al., including metabolism and replication. This unique technological achievement would lay the foundation for the production of many other kinds of synthetic cells, potentially including mirror microorganisms (see Case 8, below).

Risk Considerations

Biosafety

As JCV-syn3.0 is derived from an animal pathogen (not a human pathogen), RG1 is most appropriate for this envisioned case, although the removal of many genes from the genome likely makes the synthetic cell less robust. Although the likely human health risks would resemble those already managed for other RG1 organisms, the minimal nature of the genome and the bottom-up assembly approach may create uncertainty for IBCs in their efforts to assign an appropriate risk category.

Biosecurity

In this case the synthetic cell itself poses minimal biosecurity risk. However, the various technologies that make this cell possible could also enable the engineering of more dangerous microbial organisms. It is unclear whether the capabilities of such organisms could exceed those attainable by other engineering techniques. Any biosecurity concerns raised by this example relate more to enabling technologies in synthetic genomics than to the resulting minimal cell itself.

Ecological Considerations

This synthetic cell is unlikely to pose greater likelihood of environmental harm than the natural organism from which it is derived, M. mycoides, which is present in natural environments.

Governance Considerations

If such a system were ever achieved, its oversight in the United States would likely rely on existing policies for replicating organisms that contain synthetic DNA. Laboratory research would fall under the NIH Guidelines and IBC review, while any proposed environmental release would be handled by the federal agencies that already regulate engineered microbes, including USDA, FDA, and EPA (under TSCA, when relevant). At the same time, this case raises broader questions that current regulations do not directly address, such as how to define a living system assembled from nonliving components and whether bottom-up life should be governed in the same way as organisms derived from natural lineages, or whether its novelty may eventually call for new policy guidance.

CASE 8 – BASIC RESEARCH: MIRROR MICROORGANISM FOR CARBON SEQUESTRATION

Mirror biochemistry offers significant scientific opportunities, including insights into the chemical origins of life and the role of chirality in biological systems. Mirror biomolecules may also have valuable industrial and biomedical applications, for

example, in the production of stable materials (Schneider, 2025) or therapeutics resistant to enzymatic breakdown (Went, 2025). With the emergence of mirror biochemistry and associated research and applications, recent scientific and policy discussions have focused on the concept of mirror life, or a living organism (e.g., a microorganism) that has been built with biomolecules with the opposite chirality to those found in all known life (Adamala et al., 2024; Evans et al., 2025; NASEM, 2025; Peplow, 2025).

In theory, such mirror microorganisms would be composed of amino acids, nucleic acids, and sugars of reversed chirality. It then follows that a mirror microorganism would be expected to have the same functions as a naturally occurring equivalent, including information storage in DNA, metabolism, and replication, provided all the needed biomolecules were also mirror-image. For instance, a mirror ribosome could translate a mirror mRNA transcript, producing a chain of mirror amino acids the same way it does in a natural organism. Constructing this type of synthetic cell is expected to require assembly from non-living components. Although the synthesis of an entirely mirror-image cell is currently technologically out of reach, as is the bottom-up construction of a cell with natural chirality (see Case 7 above), proof-of-concept demonstrations in mirror-image biomolecules (Callahan et al., 2024) and synthetic cell construction have led to increased discussion of this possibility (Cai and MBDF, 2025; Ellington and Devaraj, 2025; NASEM, 2025; Zhu, 2025).

Some have made the case that mirror organisms could theoretically evade immune recognition, resist degradation by natural enzymes or viruses, and circumvent existing antimicrobial agents, leading to potential biosafety, biosecurity, and ecological concerns (Adamala et al., 2024; Epstein et al., 2025). Some scientists have also cautioned that self-replicating mirror life could establish a parallel “tree of life” beyond existing evolutionary constraints, complicating detection and control mechanisms (Zimmer, 2024). However, others have suggested counterarguments for these notions and called for more research and evidence to be collected (Congress.gov, 2025; Evans et al., 2025; NASEM, 2025; Zhu, 2025).

This hypothetical case envisions the development of a mirror-cell system based on the organism Prochlorococcus marinus MED4. P. marinus is a common ocean microbe that contributes to substantial amounts of carbon dioxide consumption and oxygen production worldwide, making the development and eventual release of a mirror version of the organism hypothetically useful as a means to sequester carbon dioxide and thereby blunt global warming. P. marinus also possesses a relatively small genome (1.66 million DNA base pairs), and would therefore require less de novo DNA synthesis and assembly for the mirror version compared to most microbes.

Choosing a photosynthetic organism as a basis for a mirror synthetic cell in principle reduces the requirements of the organism for chiral substrates, enabling the mirror cell to subsist primarily on achiral inorganic sources of carbon, nitrogen, and phosphorous. However, P. marinus can also require modest amounts of organic nutrients from its environment or from nearby helper cells, suggesting that a mirror version of this organism might not replicate as freely or as independently as intended.

Risk Considerations

Biosafety

Since this hypothetical mirror-cell system relies on recombinant and synthetic nucleic acids, research conducted at institutions under NIH oversight would fall within the scope of the NIH Guidelines. The appropriate risk group classification for a mirror bacterium, however, would depend on its demonstrated capacity to cause harm in humans. In this case, P. marinus is not considered a human pathogen. Given its role in marine environments, consideration should be given to potential impacts on animals, plants, and the broader ecosystem. However, assessing such risks is challenging because key unknowns remain about how mirror cells might interact with existing biological systems. To date, experiments needed to evaluate these interactions have not been conducted. Concerns surrounding replication-competent mirror cells have largely focused on theoretical risks, such as opportunistic infection or evasion of immune defenses, even when the original template organism is not pathogenic (Adamala et al., 2024). Yet these concerns remain hypothetical. Actual risk would depend primarily on the specific biological functions engineered into the organism rather than on molecular chirality alone. For example, in the case of mirror P. marinus, the organism’s preference for marine environments and its reliance on sunlight for photosynthesis may further limit its potential to act as an opportunistic pathogen.

Biosecurity

Mirror bacteria are still largely theoretical, and self-replicating mirror life is not something current biotechnology can produce. For that reason, there is no clear evidence of concrete biosecurity risks today. At the same time, there is active debate among scientists and policy makers about how to anticipate and govern possible future developments. Some experts argue that, if mirror organisms were ever created, they might evade natural immune defenses and environmental controls. That concern has driven calls for early governance frameworks and precautionary evidence requirements. Others see these risks as speculative at this stage. In practice, the more immediate biosecurity questions focus on the enabling technologies needed to build mirror systems and on how oversight and governance should evolve as the science progresses.

Ecological Considerations

Mirror biological systems raise novel environmental questions, but empirical data and established assessment frameworks are currently lacking. There is no evidence that self-replicating, environmentally robust mirror organisms exist today, making claims about environmental persistence or uncontrolled spread speculative. Any mirror cell would likely face strong constraints outside controlled settings. Nutrients used by natural organisms would likely be unusable in mirror cells without specialized mirror-image metabolic systems, which could limit growth in natural environments. While it has been suggested that mirror organisms may have reduced susceptibility to natural biological

threats, assessing this possibility would require experiments with a fully functional and stable mirror system, which has not been demonstrated. The primary issue is uncertainty rather than demonstrated hazard, pointing to the need for cautious, staged research and adaptive oversight as the science develops.

Governance Considerations

Given a broad spectrum of views, experts have called for proactive discussion and coordination among international scientific and regulatory bodies before technical feasibility of any kind of mirror life is achieved. Through these discussions, some have agreed that there should be a complete moratorium on efforts to create mirror life, although where to draw the line has been debated extensively and acknowledged as a potentially slippery slope (Evans et al., 2025) that could cause disruption to other beneficial areas of research, including synthetic cell research and development. Proposed approaches include careful oversight of enabling technologies, while allowing beneficial mirror biomolecule and synthetic cell research to continue under appropriate safeguards.

SUMMARY AND CONCLUSIONS

These eight cases explore how biosafety, biosecurity, and environmental considerations change as system complexity, function, and context of use evolve. The cases range from simple constructs without genetic material to complex, genome-containing, replicating entities (including mirror life), and collectively illustrate how design choices and deployment contexts shape both risk profiles and governance needs.

At the simpler end of the continuum, examples such as the chemotactic vesicle demonstrate that cell-like behaviors can emerge from purely chemical or biochemical systems that lack genomes, replication, or metabolism. At the more complex end, cases involving genome-containing, replicating, environmentally deployed, or hypothetical mirror life systems illustrate how synthetic cells may diverge from natural biology in fundamental ways, even as they retain familiar cellular attributes. Taken together, the cases reinforce that synthetic cells are not a single category of technologies or products and therefore do not present uniform benefits, risks, or governance challenges. Instead, their potential risk and benefit profiles depend on design features, intended use, and exposure pathways.

A central theme that emerges from the cases is not a simple absence of oversight, but variation in how existing frameworks apply across different synthetic cell designs and contexts. In many instances, the cases illustrate where current governance mechanisms clearly apply, where interpretation varies, and where additional guidance may be needed. Genome-free, non-replicating systems used in basic research often fall outside recombinant DNA–based policies yet still raise questions about appropriate containment and classification. Non-living but genetically encoded systems used in medical or environmental contexts challenge assumptions that link oversight primarily to living status or pathogenicity. Highly refactored organisms intended for industrial use test expectations embedded in traditional risk groups and biosafety levels. These examples

suggest that governance outcomes can differ for functionally similar technologies based on relatively small design or use-context differences, such as whether a design incorporates DNA, whether that DNA replicates, and whether the system is intended for contained use or release. This underscores the value of function- and context-based evaluation rather than reliance on categorical labels alone.

The cases also highlight ecological dimensions that are not fully captured by existing human health–centered biosafety frameworks. Notably, across the first seven cases, most synthetic cell systems would be classified as Risk Group 1 under the NIH Guidelines and Biological Safety Level 1 as described in Biosafety in Microbiological and Biomedical Laboratories (CDC and NIH, 2020), because they pose little or no direct pathogenic risk to humans. Applications involving environmental release, such as bioremediation or agricultural nutrient delivery, raise questions about persistence; interactions with native microbiota, plants, and animals; and effects on biogeochemical processes. Even non-replicating constructs can influence local environments during their period of activity or contribute DNA that is available for horizontal gene transfer. These examples suggest value in an approach that considers the health of humans, animals, plants, and ecosystems together.

From a governance perspective, the cases also reveal distribution and variability in oversight. Responsibilities shift across various agencies, including NIH, FDA, EPA, USDA, and OSHA, depending on the product claims, sector, and stage of development, which can create a patchwork of expectations for developers. The U.S. Fish and Wildlife Service may have a role to play as new environmental applications are developed and realized. At the institutional level, biosafety professionals and IBCs may reasonably reach different conclusions when evaluating similar constructs, particularly when those constructs do not fit established categories. Across the cases, the most salient governance challenges arise not from labels like “biological versus chemical” or “gain of function,” but from considering risk and benefit trade-offs, environmental hazard, and the cumulative capabilities enabled by advances in synthetic cell research.

Taken together, these cases point to several recurring needs. There are difficulties in defining and classifying systems that are non-living or genome-free but still exhibit cell-like behavior. There are gaps in ecological governance, since current systems do not provide systematic ways to assess environmental impacts. Implementation can vary significantly across institutions and agencies, and coordination challenges arise because responsibilities are distributed across the Coordinated Framework. Finally, the cases underscore the value of anticipatory governance for emerging areas, such as mirror life, where policy discussions may need to begin before technical feasibility is achieved.

Conclusion 5-1: Synthetic cells span a continuum of designs, from non-genetic biochemical assemblies to genome-containing, replicating systems. As a result, they do not constitute a single category of technologies or products, and their risks, benefits, and governance needs depend on their design features, intended use, and exposure context.