Strain Selection

In this project we used Escherichia coli Nissle 1917 (Nissle 1917) and Lactiplantibacillus plantarum WCFS1. According to DSMZ (recommended on the iGEM website), both strains are Risk Group 1 and appear on the "White List." Both have a long history of safe use in food with good safety records.

Members of Lactiplantibacillus ^[1] and Escherichia ^[2] occupy niches in the human nasal cavity; introducing these probiotics is unlikely to excessively disturb the native nasal microbiota.

In recent years, Nissle 1917 has often been engineered as a live biotherapeutic, with extensive preclinical and early clinical studies for metabolic disease and inflammatory bowel disease ^[3,4], providing evidence for human safety and preliminary efficacy. L. plantarum WCFS1 has also been widely studied as a mucosal-vaccine platform ^[5], mostly at the preclinical stage, indicating in-vivo safety.

Additionally, our work is conducted under BSL-1, with bounded dose and dosing frequency, and with multiple built-in inactivation mechanisms to further reduce risk.

The strains used here pose a low likelihood of direct harm and are considered highly safe.

Design Rationale

Our preliminary literature review indicated that engineered bacteria adhering to the olfactory epithelium (OE) may escape to other sites. We therefore designed measures to mitigate biosafety risks arising from potential escape.

Potential for Escape of Engineered Bacterias

Fig.2 Possible causes of engineered-bacteria escape

Prior studies show that while various OppA proteins bind specifically and strongly to N-acetyl-heparan sulfate (NaHS) on OE cells, the affinity is not 100% (in different cell lines, measured affinities cluster around 30--50% ^[6]). Thus, adhesion failure can occur.

Even when adhesion succeeds, engineered bacteria may still be cleared or shed due to:

The airway's natural defenses: the mucociliary clearance system is the first barrier, removing particles and pathogens and maintaining appropriate airway hydration and ionic composition ^[7].

Immune effectors and immune-cell clearance, epithelial turnover, and competition with the native OE microbiota can also cause detachment.

External factors such as sneezing or nasal irrigation may dislodge bacteria.

Given imperfect affinity and active clearance, escape from OE is plausible and must be treated as a biosafety priority.

Potential Destinations and Impacts of Escape

Fig.3 Potential destinations after escape

Escape can proceed via two main routes: to the external environment or to other internal sites. We considered destinations, survivability, and possible adverse outcomes.

The nasal cavity is open to the atmosphere and internal systems. Poorly colonizing strains may exit into the environment, raising environmental-safety concerns.

Bacteria may pass from the nasopharynx through the oropharynx, esophagus, and stomach to the gut. Although gastric acidity kills most bacteria, Nissle 1917 can maintain a neutral--alkaline intracellular pH via the glutamate-dependent AR2 system, protecting macromolecules from acid damage ^[8]. L. plantarum membranes are rich in unsaturated fatty acids, preserving fluidity and integrity in acid; H+-ATPase helps maintain near-neutral cytosolic pH. Thus, both strains have acid tolerance and could plausibly reach the intestine.

The gut hosts a dense microbiota essential for digestion, metabolism, barrier maintenance, and immune regulation ^[9,10]. If engineered Nissle 1917 or L. plantarum reach the gut, they might disturb the resident community, provoking inflammation or metabolic dysfunction and increasing the risk of diseases such as diabetes and IBD.

From the nasopharynx, bacteria can be aspirated to the lungs. The lower airway harbors a lung microbiome ^[11--13] that forms a unique ecosystem. In health, lung microbes, airway epithelium, and alveolar macrophages maintain tolerance; dysbiosis can cause disease. Normally, cough, mucociliary clearance, and macrophages remove invaders efficiently. Under conditions such as colds, smoking, or asthma, bacteria may reach bronchi or alveoli. Huang et al. ^[14] observed elevated E. coli Nissle in lung tissue shortly after intranasal dosing in mice, with inflammatory infiltration that resolved by day 7. As this risk is typically lower than the digestive-tract route, we consider it a lesser concern here.

The OE lies atop the cribriform plate, which contains foramina for olfactory axons to enter the cranial cavity. In theory, highly invasive pathogens might track along nerve sheaths retrogradely to cause meningitis or brain abscess. For our engineered strains, the intracranial entry risk is extremely low ^[6,12] and can be neglected.

Summary of Kill-Switch Strategies

Fig.4 Biosafety protection strategies

Ensuring the biosafety of engineered bacteria is paramount. To prevent harm to the environment and host, we adopt multi-layer "safety shackles." The first layer is physical containment and good microbiological practice (e.g., asepsis). The second layer is biological safeguards, i.e., built-in measures that prevent survival or function in non-target environments. Among these, kill-switch circuits are widely used by researchers and iGEM teams ^[17,18].

A typical kill switch consists of a sensor and a suicide effector. Many iGEM teams have designed creative variants, each with pros/cons. Given our needs, we summarize commonly used strategies:

Auxotrophy-based switches

Knock out genes needed to synthesize essential nutrients (amino acids, nucleotides), making strains dependent on supplemented media.

Pros: Simple, effective, hard to reverse; the knocked-out locus is selectable.

Cons: Operationally more involved; trace nutrients in the environment may enable compensatory evolution.

Inducer-dependent "ON" switches

Addition of an inducer (e.g., rhamnose, arabinose) triggers death. Without inducer, a constitutive promoter expresses a repressor that binds the toxin promoter/operator and blocks expression; with inducer, the repressor is inactivated/dissociates, the toxin is expressed, and cells die.

Pros: Highly controllable; simple, efficient trigger; inducers are non-toxic.

Cons: Performance depends on inducer dose; evolutionary escape can occur if mutants bypass the circuit.

Environment-sensor switches

Smarter circuits respond to environmental changes (temperature, light, oxygen). Temperature sensors trigger death below a set threshold; light sensors tie survival/death to illumination; oxygen sensors respond to O₂ levels (e.g., survival in normoxia, death in hypoxia, or vice versa).

Pros: Autonomous, no exogenous inducer; immediate response; suitable for open environments.

Cons: Near-threshold "leaky" toxin imposes strong selection; complex natural environments can cross-activate sensors and cause false positives/negatives.

Experimental Design of Safeguards

Based on the above, poorly adhering bacteria may escape to the external environment or gut; risks to the lung and intracranial destinations are minimal and not considered further. We compared options and selected kill-switch designs to reduce biosafety risks from these two escape routes.

External-environment escape

The nasal cavity is open. Oxygen levels inside vs. outside do not differ markedly; inducers are efficient but reduce autonomy. During breathing, nasal mucosal temperature averages 30.2--34.4 °C ^[15,16]. We idealize 37 °C at the target site and < 30 °C outside. Accordingly, we designed a temperature-sensitive system: at 37 °C bacteria grow normally; when escaping to cooler environments (< 30 °C), the system induces MazF toxin expression to kill cells.

Gut-escape

The nasal cavity is oxygen-rich, but the gut is micro-hypoxic. We therefore designed a hypoxia-responsive suicide system: in oxygen-rich OE, bacteria grow; upon reaching hypoxic gut conditions, the system activates MazF to kill cells.

Emergency containment

For contingencies, we built emergency "off" switches: in Nissle 1917, arabinose induction triggers suicide; in L. plantarum, nisin induction triggers suicide. These inducible systems minimize risk during unexpected events.

Infrastructure & Equipment

All experiments were conducted in the iGEM TJUSX laboratory at the College of Synthetic Biology and Biomanufacturing, Tianjin University. The lab complies with the Regulations on Biosafety Management of Pathogenic Microbiology Laboratories of the PRC and the Safety Code for University Laboratories, and is classified as BSL-1.

Fig.5 Laboratory Layout Functional Zoning Diagram

Our lab's functional zoning improves safety and usability. Safety-critical facilities include biosafety cabinets, clean benches, chemical fume hoods, emergency equipment (firefighting and first aid), and emergency lighting, which together protect personnel.

Safety Training

To ensure safety, we trained every participant on lab rules and conduct regular safety assessments. Core rules include:

General principle: Safety first, with prevention foremost.

1. General conduct
   1. No entry without authorization and completed safety training.
   2. Before any experiment, understand materials' physicochemical properties, hazards, and emergency measures.
   3. Keep areas, benches, and floors clean, unobstructed, and orderly; do not block aisles or emergency equipment.
2. Prohibited behaviors
   1. No eating, drinking, or smoking in lab areas.
   2. No horseplay or non-experimental activities.
   3. Do not wear lab coats or gloves into offices, lounges, cafeterias, or public spaces.
   4. Wash hands thoroughly with soap and water before leaving. Replace/clean lab coats after experiments.
3. Personal protective equipment (PPE)
   1. Wear lab coats; minimize exposed skin. Remove lab coats when leaving. Avoid touching public surfaces (door handles, phones, keyboards) with gloves.
   2. For hazardous tasks, wear appropriate safety glasses/goggles.
4. Chemical safety
   1. Label all reagents with name, date, responsible person, and hazard warnings; inspect regularly and replace when needed. Use and store strictly per regulations.
   2. Follow the "three no's": do not smell, taste, or directly touch any reagent.
5. Equipment safety
   1. Do not operate large/precision/high-risk instruments (e.g., autoclaves, centrifuges) without training and authorization.
   2. Check integrity before use (cables, plugs, guards).
   3. Follow SOPs; stop and report any abnormality.
   4. After experiments, turn off unused instruments and shut off water/gas/power (except for required continuous-run equipment like refrigerators).
6. Emergencies & response
   1. All personnel must know emergency exits/escape routes, safety showers/eyewash stations (test weekly), fire extinguishers, and first-aid kits, and be familiar with emergency procedures.
   2. Report any incident or anomaly immediately, no matter how small, to the lab safety lead for investigation and prevention.

Waste Disposal

Waste handling is critical for personnel and experimental safety. After each session, a duty roster ensures timely collection. A two-round check by different personnel verifies toxicity and classification before disposal.

Risks in the Laboratory

We use highly safe probiotics (Nissle 1917 and L. plantarum) and build suicide circuits, greatly reducing biosafety concerns. Still, accidental release is possible. We therefore enforce strict supervision, periodic audits of practices and biosafety measures, routine biosafety training, and emergency drills to minimize risk.

Potential Application Risks

Direct health risks to patients

If nasal-colonizing engineered bacteria are used therapeutically, we must consider risks such as inflammation, hyper-immune responses, and off-target effects. Clinical deployment requires multiple rounds of evaluation and trials to mitigate impacts.

Environmental & public-health risks

After treatment, patients may release engineered bacteria into the environment (e.g., via sneezing or rhinorrhea), and long-term ecological effects are uncertain. We will conduct deeper investigations, including expert interviews, and proactively address potential impacts.

Ethical & societal risks

Public concerns may cause psychological resistance to engineered probiotics. We therefore enhance outreach via online platforms (e.g., WeChat) to improve understanding and acceptance.