Safety

Genetic Modifications for Safety

YES302

Chassis & Core Modification: Uses Escherichia coli Nissle 1917 (EcN, an iGEM-recognized safe chassis). The asd gene (encoding diaminopimelic acid synthase) is knocked out via λ-Red recombination, rendering the strain DAP (diaminopimelic acid)-auxotrophic. A plasmid harboring asd (to complement the δasd defect) and XanQ (with a F94Y88 point mutation to enhance xanthine transport) is introduced.

Safety Mechanism: Employs a balanced-lethal system. The strain survives only in DAP-supplemented media or when pYES302 is present, preventing environmental colonization.  Verification: When inoculated into LB medium without DAP, YES302 showed OD₆₀₀ < 0.05 (no growth); in DAP-supplemented LB, OD₆₀₀ reached 1.2, confirming DAP-dependent survival.

YES303

Gene Source & Function: Expresses UacT (a uric acid transporter) from E. coli K-12 MG1655.  UacT exclusively mediates uric acid transmembrane transport with no virulence-related functions.

Functional Verification: Colorimetric assay kit detection showed YES303 reduced uric acid concentration from 200 μM to 85 μM in simulated intestinal fluid (pH 7.0) within 2 hours, while empty-vector EcN only reduced it to 180 μM, confirming transport activity without side reactions.

YES304

Truncated Uricase Design: Expresses truncated UricaseT (deletion of N-terminal 1–169 amino acids, non-catalytic domain), reducing the protein molecular weight from 55 kDa to 36.7 kDa while retaining the catalytic domain for uric acid degradation.

Secretion & Activity Verification: Western Blot detected UricaseT in culture supernatants (partial secretion). The spectrophotometric absorbance results  showed that after 4-hour co-incubation with 1 mM uric acid, YES304 reduced residual uric acid to 120 μM, while empty-vector EcN left 980 μM, confirming enzymatic activity.

Horizontal Gene Transfer Verification

Experimental Design: YES302 was serially passaged in LB medium every 12 hours  for 20 generations.

Results:

Plasmid Retention: Colony PCR of 10 random colonies per generation showed 100% plasmid retention over 20 generations.

Functional Stability: HPLC measured xanthine transport efficiency of YES302 at generation 0, 10, and 20 in simulated intestinal fluid. Transport rates for 100 μM xanthine were 92%, 90%, and 88%, respectively (no significant difference, p > 0.05), confirming stability.

Laboratory Safety Protection Equipment

Biosafety cabinet

Hazardous Chemical Cabinet

Laboratory Attire & Basic Protocols

Mandatory Attire:

Lab Coats: Long-sleeve coats must be worn. Coats are UV-sterilized for 30 minutes post-use and stored at the lab entrance.

Gloves: Nitrile gloves (not latex, to avoid allergies and microbial penetration) are mandatory for handling engineered bacteria/hazardous chemicals. Gloves are changed immediately if torn/contaminated, using no-touch removal to avoid wrist contamination.

Goggles: Required for centrifugation (≥10,000 rpm), hazardous chemical pouring (e.g. HCl, acrylamide), and aerosol-generating tasks (e.g. bacterial sonication). Anti-fog goggles ensure clear visibility.

Prohibitions & Good Practices:

No eating/drinking/storing personal items (phones/snacks in labeled lockers at the lab entrance). No makeup/jewelry (rings/bracelets) in the lab. Long hair must be tied back and tucked into lab coats to prevent reagent contact or entanglement in equipment (e.g. centrifuges, shakers). Post-experiment, workbenches and biosafety cabinet interiors are wiped with 75% ethanol. Lab coats are stored in designated areas and not taken outside (laundered via university-approved sterilization services if needed).

Engineered Bacteria Manipulation SOP

Gene Editing Safety:

λ-Red Recombination & Cre-loxP System: All gene editing occurs in Class II biosafety cabinets. Cabinets are ventilated for 30 minutes pre-operation, with the sash kept ≤15 cm above the work surface.

Temperature-Sensitive Plasmid Curing: Plasmids like pKD46 (30°C replication, 42°C inactivation) and pKD-Cre are cured by incubating strains at 42°C for 2 hours, followed by 3 passages at 37°C. PCR verification  confirms no residual plasmids.

Gastrointestinal Simulation Experiments:

Simulated Fluid Preparation: Simulated gastric fluid and simulated intestinal fluid are filter-sterilized (0.22 μm).

Operation: YES302 (OD₆₀₀=1.0) is mixed 1:1 with SGF (37°C, 5 min), then 1:1 with SIF (37°C, 60 min). All steps use screw-cap centrifuge tubes sealed with parafilm to prevent leakage/aerosols. Post-experiment, contaminated consumables (tubes, pipette tips) are autoclaved (121°C, 20 min).

Safety Training

Hands-On Training:

Biosafety Cabinet Operation: Under the training of Senior Lab Technician Zhang (Biosafety Officer of East China University of Science and Technology, ECUST), the team mastered the skills of sterile plating and pipetting for plasmid-transformed cultures in a biosafety cabinet. The team’s operational competence was verified through post-plating observation: no contaminant growth was detected, which confirms proficiency in maintaining sterility during the operation.

Autoclave Use: The team received training on the key operational procedures of autoclaves, including program setting, pressure/temperature calibration, and post-sterilization venting. The effectiveness of the sterilization process was validated using biological indicators: Geobacillus stearothermophilus spore strips. After the sterilization treatment, no color change was observed on the spore strips, indicating that the sterilization process met the required standards.

Hazardous Chemical Management

Storage & Access:

Categorized Storage:

Corrosives (1 M HCl, 1 M NaOH): Stored in acid/alkali-resistant cabinets (LABCOCO C1000) with leak-proof trays.

Flammables (anhydrous ethanol, acetone): Stored in explosion-proof refrigerators.

Toxins (acrylamide, SDS): Stored in double-locked cabinets, with keys held by PI + safety officer (dual-signature log for access).

MSDS Archiving: Electronic MSDS are stored at E:\LabSafety\MSDS_2025; printed copies are posted near reagent cabinets for emergency access (e.g. HCl spill response).

Handling Protocols:

1 M HCl: Used in a fume hood (goggles + nitrile gloves). Spills are neutralized with 0.1 M NaOH, cleaned with absorbent paper, then rinsed with water.

Acrylamide (Neurotoxin): Weighed in biosafety cabinets with double nitrile gloves (thin inner, thick outer). Waste acrylamide is collected in dedicated bottles and not mixed with other waste. Hands are washed thoroughly with soap post-operation.

High-Risk Experimental Procedures

Gene Editing Risks (λ-Red Recombination): Centrifugation (12,000 rpm, 4°C, 2 min) poses aerosol risks. Sealed centrifuge tubes (Eppendorf Safe-Lock) are used; tubes stand for 5 minutes in biosafety cabinets before opening. Workbenches are sprayed with 75% ethanol, and cabinets run for 15 minutes post-operation.

GMO Containment & Waste Disposal

Biological Waste:

Engineered Bacteria Waste: Consumables (petri dishes, pipette tips, centrifuge tubes) and cultures contact with GMOs are placed in red biohazard autoclave bags, sealed, and autoclaved (121°C, 20 min, 103.4 kPa) before disposal as general solid waste.

Environmental Risk Mitigation

Chemical Waste:

Categorized Collection: Organic (acetone, ethanol), inorganic (HCl, NaOH), and nitrogen-containing (acrylamide) waste are stored in labeled bottles ((label content: component, concentration, collection start date, estimated volume). A licensed company collects waste quarterly, with transfer documents archived.

Human Practices Data Privacy

Surveys & Interviews:

Anonymization: Surveys on “hyperuricemia patient lifestyles” use questionnaire numbers  (SKLBE-2025-001 to 200)  instead of personal identifiers. Sensitive info (age, disease duration) uses ranges (e.g. 30–40 years old, 1–3 years).

Informed Consent: During hyperuricemia-related information collection (e.g. lifestyle surveys, interview records), the team strictly follows personal information protection principles: for minors, if portrait rights or personal health information are involved, the team first details the research purpose (needs assessment for probiotic-assisted hyperuricemia treatment), information usage (iGEM-only, no commercial use), storage (encrypted, destroyed 1 month post-project), and right to withdraw to their legal guardians, and only collects information after obtaining guardians’ explicit permission; for adults, the team communicates directly with them to clarify the scope of personal information collection, usage boundaries, and safety measures, and proceeds with collection only after confirming their acknowledgment of all information processing matters. Notably, the team did not sign written agreements or contracts with participants, but retained communication evidence (e.g. oral confirmation records, electronic/paper information notification receipts) to ensure all collection is based on participants’ or guardians’ explicit approval, aligning with core personal information protection requirements.

Data Destruction: Paper surveys are shredded (≤5 mm×5 mm) by two team members; electronic data is wiped via  DBAN  under university IT supervision, with a  Data Destruction Confirmation Form  archived.

Expert/Patient Interviews:

Recording Authorization: For expert/patient interviews requiring recording or videoing, the team communicates with interviewees via WeChat: we clearly inform them of the intended use (for iGEM Wiki/posters after anonymization), respect their wishes, and obtain their explicit consent. Notably, no written Content Use Authorization Forms are signed, but all recording/videoing activities are based on interviewees’ clear approval.

Content Processing: Transcripts are sent to interviewees for approval; identifiers (e.g. “Dr. Zhang, Endocrinology, Shanghai XXX Hospital”) are anonymized (e.g. “Endocrinologist at a tertiary hospital”).

Communication Records

iGEM Correspondence: Emails with the iGEM Safety Committee (e.g.  Animal Use Form  Q&A, Safety Check-In Form approvals) are archived as PDFs ( AnimalUseForm_QA_20250910.pdf ,  CheckInForm_Approval_20250820.pdf ) in an encrypted Baidu Cloud folder (access code managed by the safety officer).

Internal Communication: Weekly safety meeting minutes and risk assessment reports are archived to ensure traceability of all safety decisions.

Communication Records with iGEM

The team submitted the Animal Use Form to the iGEM Safety Committee on August 13, 2025, detailing:

Mouse strain selection: 5-week-old male C57BL/6 mice (from Ziyuan, Hangzhou) were chosen—this inbred strain has a stable genetic background (ensuring result reproducibility), is widely used in metabolic disorder studies, and male mice avoid hormonal fluctuations interfering with uric acid metabolism.

Animal welfare safeguards: Mice had a 1-week acclimation period, were housed in a controlled environment (22±2℃, 65% humidity, 12h light-dark cycle) with standard diets; operations (e.g. gavage, non-invasive feces collection) were standardized to reduce pain; health was monitored (experiment terminated for abnormalities); euthanasia followed AVMA guidelines, and the 3R principles were adhered to.

Laboratory qualifications: Experiments were conducted at East China University of Science and Technology’s Experimental Animal Center, with protocols approved by the university’s Animal Care Committee (No. 2006272), following China’s MOST-2011-02 guidelines and institutional animal ethics approval.

iGEM Q&A Process: After receiving the reply from the iGEM Safety Committee, we promptly revised and improved the content of the Animal Use Form. We provided explanations for the previous content and added text related to YES302. In the original text, the reasons for using only male mice in the experiment were not strongly elaborated. Now, we have listed several other similar projects that also used only male mice in their mouse experiments, which demonstrates the rationality of our model. In addition, we further specified the source of the mice. We detailed the method for euthanizing the mice and listed the Humane Endpoint indicators. Finally, we also included the description of the mouse experiment design for YES302. By the iGEM submission deadline (October 5, 2025), although no final approval response was received, the team completed 3 rounds of Q&A communication with the iGEM Safety Committee (email records archived as iGEM_AnimalUse_QA_20250910-1005.pdf).

Future Safety Prospects

Conduct survival experiments on YES302 in soil and water (simulating the scenario of accidental release of experimental animal excreta).

Detect viable cell counts and gene residues (qPCR detection of XanQ) at different time points to enrich environmental safety data.