All experimental work was conducted in the certified BSL-1 laboratory facility of BPGbio located in Boston, in full compliance with the iGEM Safety and Security Policy. Prior to entering the laboratory, each team member was required to complete onsite biosafety and laboratory safety training organized by BPGbio. The training program covered fundamental laboratory safety protocols, proper use of personal protective equipment (PPE), biohazards, chemical hazards, waste disposal procedures, and emergency response protocols. Additionally, all team members participated in laboratory orientation tours led by Senior Facility Manager Mr. Ken Epstein.

PPE must be worn at all times when entering the laboratory. BPGbio-provided PPE includes lab coats, nitrile gloves, and safety goggles. All team members are also required to wear long pants, closed-toe and flat shoes, and to tie back long hair. Additional task-specific protective measures are implemented as needed. For example:

• Heat-resistant gloves are provided during PCR setup;
• UV face shields and double gloves are required when performing gel electrophoresis;
• Face shields, safety goggles, aprons, and gloves are worn when handling strong chemical reagents during gel extraction;
• During DNA ligation or ligation reactions, all microcentrifuge tubes must remain on ice and securely capped to prevent DNA leakage;
• In transformation and electroporation experiments, all electroporation cuvettes and gloves must be completely dry to minimize the risk of electrical shock;
• Insulated gloves are provided during Western blotting procedures to safely handle warm transfer apparatus.

Emergency eyewash stations and safety showers are accessible in every laboratory room and corridor. All equipment is capable of delivering continuous water flow for at least 15 minutes. Any exposure incident involving corrosive or hazardous substances must be reported immediately.

All live procedures are performed within a Class II, Type A2 biological safety cabinet (BSC). Laboratory work surfaces and equipment are decontaminated before and after use with either 70–75% ethanol or bleach solution containing 0.5–1.0% available chlorine. Bacterial strains are stored in locked -80°C ultra-low temperature freezers and transported in sealed, leak-proof containers to prevent accidental release.

Figure 1. Laboratory Safety Facilities

The engineered bacterial chassis used in this project is Escherichia coli Nissle 1917 (E. coli Nissle 1917), which belongs to Risk Group 1 (RG1) under the biological safety classification system. This strain has been safely used for decades as a probiotic (e.g., Mutaflor®) in human clinical applications, with no documented pathogenicity or environmental persistence.

To enable neohesperidin production, eight genes were introduced into E. coli Nissle 1917. Prior to plasmid construction, we verified the origins of all genes used. All genetic components are listed on the iGEM-approved whitelist.

Gene Abbreviation	Full Name	Source Organism
ThF3′H	Flavonoid 3′-hydroxylase	Tricyrtis hirta
CPR	Cytochrome P450 reductase	Arabidopsis thaliana
VvRHM-NRS	UDP-4-keto-L-rhamnose reductase RHM1	Vitis vinifera (wine grape)
UGT73B2	UDP-glycosyltransferase 73B2	Arabidopsis thaliana
Cm1,2RhaT	Citrus maxima flavonoid 1,2-rhamnosyltransferase (C12RT1)	Citrus maxima
CadC	Transcriptional activator CadC	Escherichia coli (strain K12)
MazF	Endoribonuclease toxin MazF	Escherichia coli (strain K12)
MazE	Antitoxin MazE	Escherichia coli (strain K12)

All genes are derived from non-pathogenic organisms and are not associated with virulence, antibiotic resistance, or horizontal gene transfer risk.

We have designed a dual-environment-dependent suicide switch based on a logic-gated genetic circuit, integrating two orthogonal environmental sensors to ensure strict containment under both clinical and environmental conditions. This system drives the expression of MazF, a sequence-specific endoribonuclease toxin that cleaves mRNA and triggers rapid cell death.

1. In Vivo Safety: Arabinose-Induced Suicide

To enable human-controlled termination of the engineered strain during clinical application, we employ the arabinose-inducible pBAD promoter to drive mazF expression. In the absence of arabinose, the AraC repressor protein prevents transcription, ensuring stable bacterial growth. Upon oral administration of L-arabinose (a non-toxic, FDA-approved sugar), the inducer binds AraC, triggering strong and rapid expression of MazF.

Experimental Validation:

We performed in vitro killing assays under induced conditions. When E. coli Nissle 1917 carrying the pBAD::mazF circuit was exposed to 0.2% L-arabinose for 4 hours, cell viability dropped by >99.9% (from 10⁹ CFU/mL to <10⁶ CFU/mL), as quantified by colony-forming unit (CFU) counts. Microscopy confirmed widespread cell lysis and membrane disruption. This demonstrates rapid, complete, and controllable bacterial clearance upon demand — a critical safety feature for patient protection.

This system provides human-controlled safety: If adverse effects arise, oral arabinose can be administered to eliminate the engineered strain within hours, ensuring clinical reversibility and patient safety.

2. In Vitro / Environmental Safety: Cold-Induced Suicide

To prevent environmental dissemination via fecal excretion, we employ the cold-inducible pCspA promoter — a natural E. coli stress response element activated below 20°C. Under physiological conditions (37°C, human gut), pCspA remains completely repressed. However, upon excretion into the external environment — where ambient temperatures fall below 20°C (e.g., in sewage, soil, or water) — pCspA becomes highly active, triggering MazF expression and cell death.

Figure 2. Safety System

Experimental Validation:

We incubated pCspA::mazF strains at 37°C (simulating gut) and 16°C (simulating environment). After 24 hours:

• At 37°C: >95% cell viability maintained (no MazF expression);
• At 16°C: >99.8% cell death (CFU reduced from 10⁹ to <2×10⁶). RNA-seq confirmed >50-fold upregulation of mazF transcript at low temperature.

This system ensures environmental self-destruction: Even if engineered bacteria exit the body via feces, they cannot survive in ambient temperatures and will die before reaching natural ecosystems, preventing gene flow or ecological impact.

Synergistic Design

These two systems work independently yet complementarily:

• Arabinose = Human-initiated kill switch (for clinical safety);
• Cold = Environment-triggered kill switch (for ecological safety).

Together, they form a double-lock mechanism that ensures the strain:

• Survives and functions only in the warm, arabinose-free human gut;
• Dies immediately if exposed to arabinose (patient safety) or cold (environmental safety).

Additional Assurance

Currently, all experiments are strictly limited to in vitro studies and do not involve human testing, environmental release, or vertebrate animal experiments. Such activities will not be conducted without prior institutional biosafety review and regulatory approval.