Table of Contents

• Overview
• 1. Our Project & Scope
• 2. Governance & Approvals
• 3. Intrinsic Biosafety by Design
• 3.1 Genetic-level Safeguards
• 3.2 Payload-level Safeguards
• 3.3 Material-level Self-Limiting Features
• 4. Operational Controls
• 5. AMR Risk: Response to Reviewer
• 6. Chemicals & Cyberbiosecurity
• 7. AI Tools Risk
• 8. Future Use & No-Release Policy
• 9. People & Contacts
• 10. Ethics & Human Practices
• 11. Documents & Evidence

Overview

Our project designs a DNA origami container carrying CRISPR-Cas9to specifically target themecAgene responsible for methicillin resistance in Staphylococcus aureus (MRSA). We test feasibility only in safe laboratory E. coli models to explore whether such a strategy could reduce antibiotic-resistance phenotypes in the future.

• What we do: Load sgRNA/Cas9 into DNA origami, evaluate in-vitro mecA cleavage, and use safe cellular surrogates (e.g., lacZ) for any in-cell tests.
• Boundaries: No clinical isolates, no human/animal experiments, no release beyond containment. All work occurs in authorized BSL-1 labs and inside biosafety cabinets (BSC).
• AMR risk mitigation: Dual layer genetic design to avoid HGT + strict operational containment.
• Governance: iGEM Safety Form registered; IBC approval under Zhejiang University-University of Edinburgh Joint Institute (ZJE) teaching center rules.
• More compliance details: See Safety Form (submitted and passed initial review; includes lab photos, training & facility info, and materials lists).

1. Our Project & Scope

Standard laboratory strains used:

• E. coli ATCC 25922 — used for membrane-permeabilization assays
• E. coli MG1655 — used for blue-white selection

AMR template strictly in vitro:

• The mecA gene was PCR-amplified from a purchased plasmid and used only as an in-vitro DNA template for cleavage validation.
• We do not express mecA in cells. For cellular-level validation, we use a safe reporter gene (lacZ) as the replacement target.

Not included:

• No human/animal experiments
• No clinical isolates
• No Risk Group 3/4 pathogens
• No unverified environmental or commercial sources

More details: Safety Form 3, 12, 17.

2. Governance & Approvals

• iGEM compliance: We follow iGEM Prohibited Activities, White List, AMR Policy, and Release Beyond Containment guidelines. Because some of our planned work involves materials not covered by the White List, we submitted a Check-In Form to the iGEM Safety and Security Committee and received approval before starting.
• Institutional review: We applied to use the ZJE Biomedical Teaching Center seminar lab (D501) under the open-use policy. The project is supervised by a PI, and IBC approval was obtained. Per institutional requirements, there are regular IBC inspections and PI supervision. Every student involved in wet-lab work signed a safety commitment form. Any change to the experimental plan is re-submitted for record and approval.
• Training & records:
• All team members completed laboratory safety training and passed the Biomed-X Laboratory Safety Exam, which covered PPE usage, waste management, biosafety levels, equipment operation, and emergency response.

Figure. Example page from the Biomed-X Laboratory Safety Exam, which all team members completed as part of mandatory safety training.

• Work was performed on open benches in a BSL-1 laboratory, which is appropriate for the organisms and parts we used.

Figure. Sterile filtration of TAE/Mg²⁺ buffer performed at a clean bench, illustrating proper aseptic technique in a BSL-1 laboratory.

• SDS are available for all reagents.
• Daily experiment records are maintained.

More details: Safety Form 2, 24-27.

3. Intrinsic Biosafety by Design

Our system layers genetic, payload, and material-level safeguards so function only occurs under narrowly defined, lab-controlled conditions, minimizing risks of HGT, unintended activation, and persistence outside containment.

3.1 Genetic-level Safeguards (chassis & vectors)

De-mobilization (no conjugation):

• Plasmid backbones lack transfer determinants (e.g., oriT/tra/mob), preventing conjugation.
• Constructs encode no integrases/transposases; no genomic integration functions.
• Only involve plasmids in experiments in vitro; no risk of conjugation or genomic integration during the experimental process.

Restricted host range (narrow replication):

• Replicons are limited to laboratory E. coli; replication is not maintained in unrelated environmental or pathogenic strains.
• Preference for non-broad-host-range origins further restricts maintenance.

Single-use scope (no clinical amplification):

• All cloning/validation confined to BSL-1 E. coli.
• No propagation in clinical isolates or environmental samples.

Sequence-level targeting hygiene:

• In cells we never target AMR genes; we use safe reporters (e.g., lacZ).

3.2 Payload-level Safeguards (DNA origami CRISPR)

3.2.1 Locked-then-unlock origami container:

• Protection before entry: sgRNA/Cas9 are physically shielded inside the origami, reducing extracellular exposure and proteolysis.
• Intracellular unlocking (dual-key): Gate 1SH cleaves disulfides; Gate 2: RNase H cuts engineered RNA-DNA hybrids to release sgRNA/Cas9 only after Gate 1.

3.2.2 Aptamer targeting & local enrichment:

• Origami displays DNA aptamers recognizing bacterial surface motifs, enriching payload at the correct cells.
• NPN uptake assay: aptamer-origami-G4/hemin (DO^A_PAMGH) shows higher fluorescence than non-aptamer controls (DO_PAMGH) — local permeability enhancement.

Figure. NPN uptake assay comparing DO_PAMGH and DO^A_PAMGH. Aptamer decoration significantly increases RFU values, indicating enhanced membrane permeability.

Figure. NPN uptake factor analysis showing DO^A_PAMGH has higher uptake efficiency than DO_PAMGH, confirming aptamer-mediated enrichment.

• Confocal microscopy: DO^A_PAMGH yields significantly stronger fluorescence around E. coli than DO_PAMGH controls — validating targeting effect.

3.2.3 Membrane permeabilization via G4/hemin DNAzyme (self-limiting):

• Origami-tethered G4/hemin (DO_PAMGH) amplifies peroxidase-like activity, aiding transient permeabilization.
• ABTS assay: DO_PAMGH shows higher absorbance at 415nm than free G4/hemin.

Figure. ABTS assay showing that DO_PAMGH has significantly higher peroxidase-like activity than free G4/hemin or hemin alone, confirming catalytic amplification.

• NPN uptake: DO_PAMGH > buffer; trends with EDTA positive control indicate designed outer-membrane disruption.

Figure. NPN uptake assay (RFU) comparing buffer, cells, EDTA, and DO_PAMGH. DO_PAMGH significantly increases membrane permeability, consistent with the EDTA positive control.

Figure. NPN uptake factor analysis under the same conditions, further confirming that DO_PAMGH enhances outer-membrane permeability compared with controls.

• Self-limitation: Requires hemin + H₂O₂ away from assay microenvironments, activity decays rapidly.

3.2.4 Internalization as a hard gate:

• Confocal imaging confirms intracellular signal with DO^A_PAMGH; DO_PAMGH controls are markedly lower.
• No internalization → no GSH trigger → no RNase H release → no CRISPR cleavage.

3.2.5 Cleavage validation without AMR spread risk:

• In vitro only for mecA: mecA is PCR-amplified from a purchased plasmid and used solely as an in-vitro DNA template; never expressed in cells.

Figure. In-vitro cleavage of PCR-amplified mecA fragment by sgRNA/Cas9, indicating the mecA gene was successfully cleaved.

• In-cell surrogate: functional cutting was demonstrated using lacZ in E. coli MG1655, visualized via a blue-white selection. Successful Cas9 cleavage converted blue colonies to white, confirming intracellular function without involving AMR genes.

Figure. Cleavage of lacZ reporter fragment in E. coli MG1655, used as a safe surrogate for in-cell validation.

Figure. Blue/White selection result. A. Numerous blue colonies were observed in the control group. B. Both blue and white colonies were observed in the experiment group treated with the origami platform.

• Assay pairing: membrane-permeabilization tests in E. coli ATCC 25922; reporter cutting in E. coli MG1655.

3.3 Material-level Self-Limiting Features

• Nuclease & temperature sensitivity: DNA origami degrades under DNase and during prolonged 37°C incubation — natural fail-safe limiting persistence.
• Cofactor dependency: G4/hemin activity strictly requires hemin/H₂O₂; without both, permeabilization does not proceed.
• Post-release decay: Packaging protects Cas9 pre-entry, but after release Cas9 is subject to intracellular proteolysis, limiting lifetime.
• Non-living carrier: DNA origami is non-replicating — no growth/colonization outside controlled experiments.

4. Operational Controls

• Laboratory level: all work was conducted in authorized BSL-1 facilities using open-bench practices suitable for Risk Group 1 organisms. No BSC was required, since we did not handle pathogens, clinical isolates, or Risk Group 2+ materials.
• PPE: lab coats, gloves, goggles; no food/drinks or unrelated items.
• Waste disposal: all cultures and DNA materials autoclaved or chemically inactivated and disposed of as biomedical waste.

Figure. Disposal of used syringe needles into a sharps container, demonstrating safe handling of sharp laboratory waste.

Figure. Autoclave used for sterilization of biological materials.

Figure. Sterilization of liquid laboratory waste prior to disposal, ensuring all effluents are inactivated.

Figure. Segregation of ordinary trash and biological waste. Biological waste is collected in yellow medical-grade bags and handed over to licensed waste disposal companies.

• Inventory & procurement management:
• All strains, plasmids, and reagents were procured via the official Zhejiang University ordering system, ensuring traceability and institutional oversight.
• Procurement logs confirm that mecA was obtained only as a commercial plasmid (for PCR template use only), and that all bacterial strains used (e.g., E. coli MG1655, ATCC 25922) are laboratory-safe strains.

Figure. Record from the institutional procurement system, showing traceable purchase of laboratory-safe E. coli strains and commercial plasmids.

More details: Safety Form 2, 25-27.

5. AMR Risk: Response to Reviewer

Reviewer concern: Ensure mecA is not horizontally transferred.

Our measures:

1. No mecA expression in clinical or pathogenic strains (indeed, never in cells).

2. Non-mobilizable plasmids lacking conjugation elements.

3. Workflow containment: all experiments were performed under BSL-1 containment using open-bench practices; all waste was sterilized (autoclave/chemical inactivation), and no live material left the lab.

6. Chemicals & Cyberbiosecurity

• Chemicals: handled under SDS-based training (e.g., ethanol, Ni-NTA); fire protection and spill-prevention procedures in place.
• Cyberbiosecurity: DNA/protein sequences stored on secured institutional servers with access control; no uncontrolled online sharing of engineered sequences.

7. AI Tools Risk

• AI tools used for structure prediction and sequence assessment (e.g., AlphaFold/Rosetta as applicable).
• No generative AI used to design novel risky sequences; all outputs undergo manual review by dry-lab and PI.
• No unverified sequences synthesized; only safe reporters or trusted-supplier fragments used.

More details: Safety Form 14.

8. Future Use & No-Release Policy

• Competition stage: laboratory in-vitro validation only; no in vivo or environmental release.
• Hypothetical future: any animal/clinical step would require ethics review and compliance with applicable drug-regulatory pathways; this page is research documentation, not medical advice.

More details: Safety Form 16-17, 23.

9. People & Contacts

• Team & Responsibility: Principal Investigator (PI), Laboratory Safety Officer (BSO/EHS), and Team Safety Lead — all information is consistent with our Safety Form submission.
• External support and reporting pathway:
• If a new risk is identified, the team will first conduct internal self-assessment,
• then report to the PI and lab safety officer,
• and finally escalate to the Institutional Biosafety Committee (IBC) for review if necessary.

More details: Safety Form 21.

10. Ethics & Human Practices

• No human or animal samples were used in our experimental work.
• Any future clinical applications would require ethics committee approval, informed consent, and strict data anonymization.
• For Human Practices activities (surveys, interviews):
• Participation is voluntary; participants may withdraw at any time.
• No personally identifiable information (PII) (e.g., name, phone, address, email) is collected.
• All responses are anonymized, used only for academic purposes, and stored in encrypted environments.
• Data will be deleted at the end of the iGEM season (Dec 2025) or permanently anonymized if used for long-term display.
• Participants have the right to access, modify, or withdraw any of their contributed data.
• All practices follow GDPR standards and iGEM privacy policies.

11. Documents & Evidence

• Submitted & approved: Preliminary and Final Safety Form (includes lab photos, facilities, PPE, training evidence, waste-management workflows).
• Updates: this page and the Safety Form will be continuously updated with new results and changes.