Overview

At Stanford iGEM, safety is central to responsible synthetic biology. Our project involves creating a dual RADAR system, targeting specific biomarkers associated with Hepatitis B Virus-driven hepatocellular carcinoma (HBV-HCC). While this has therapeutic potential in the long term, during iGEM our work is restricted to proof-of-concept experiments in safe laboratory conditions. Below, we outline how we designed our project with safety in mind and how we practice safe research.

Safe Project Design

Non-pathogenic chassis

All DNA cloning and plasmid preparation is performed in E. coli strains DH5-α and DH10B, which are non-pathogenic and contain multiple auxotrophies limiting survival outside the lab.

Mammalian experiments are performed only in established human cell lines (HEK293T and Hep3B) for transient transfection. These cells cannot survive outside sterile conditions and cannot spread autonomously in the environment.

Avoiding infectious agents

We do not work with live viruses, pathogens, or primary human/animal samples.

While Hep3B cells contain integrated fragments of HBV, they cannot produce infectious virus particles.

Safe therapeutic output

Our system is designed to produce a modular therapeutic protein (a split IL-2 mimetic). While high-dose IL-2 therapy can be toxic in clinical contexts, our proof-of-concept experiments plan to only involve controlled expression in cell culture.

Safe Lab Work

Workspaces

BSL-1: Open bench space for E. coli cloning and transformations.

BSL-2: Tissue culture room with biosafety cabinets for handling mammalian cell lines. Standard PPE was used at all times.

Daily safety practices

• Wearing PPE (lab coats, gloves, goggles) at all times.
• Using biosafety cabinets for all mammalian cell work.
• Decontaminating work surfaces and liquid waste with ethanol and bleach.
• Proper segregation and autoclaving of biological waste before disposal.

Hazardous Materials

Hazardous chemicals managed carefully:

• Ethanol (flammable) – handled away from open flames.
• Trypsin (irritant) – used with eye protection and gloves.
• DMSO (flammable, absorption risk) – kept in small aliquots and handled under fume hood when possible.
• Sodium pyruvate & TAE buffer (irritants) – handled with gloves, waste disposed safely.
• Sodium azide (toxic) – used only in small quantities for FACS buffer and disposed in compliance with hazardous waste protocols.

Cell Line Safety

Hazards from cell lines

HEK293T: immortalized cell line with adenovirus fragments, not infectious.

Hep3B: liver cancer line with integrated HBV sequences; cannot produce virus, negligible infection risk.

Both cell lines are handled exclusively at BSL-2 under strict containment.

Future Risks

If developed into a therapeutic, our sensor-actuator system would function inside the human body as a therapeutic platform:

• Dual-use concerns – knowledge of modular dual-gated systems could theoretically be misapplied, though our designs are built only for therapeutic use.
• Equity considerations – precision therapies may initially be costly and inaccessible.

We have openly discussed these risks within our team and with external advisors through Integrated Human Practices (IHP), ensuring ethical reflection alongside technical development.

We have also hosted a bioengineering program over the summer for high schoolers and plan to share our biosafety practices, ensuring that the next generation of researchers is trained with the same rigor.

Risk Management

Training & rules

All team members completed Stanford EH&S biosafety and chemical safety training, delivered by the UTL lab manager Mong Saetern.

We follow the NIH Guidelines, CDC BMBL, and Stanford's Biosafety Manual.

Risk management systems

• Accident reporting protocols.
• Physical access controls for lab spaces.
• Strict inventory controls for plasmids, chemicals, and reagents.
• Medical surveillance protocols if unexpected exposures occur.

Additional precautions

• No release of GMOs or products outside containment.
• No human subjects research without formal review/approval.
• Regular consultation with biosafety officers and faculty mentors.

Expert Support

We work closely with:

Faculty mentors:

Prof. Drew Endy, Prof. Xiaojing Gao, Prof. Renupathy Dhanasekaran, Prof. Huijun Ring, Dr. Philip Kyriakakis, Dr. Alex Engel, Cyrus Knusden, Samuel King

iGEM Student mentors:

Heloise Hoffmann, Alice Finkelstein, Julia Vu, Ayushi Mohanty

Lab managers & biosafety officers:

Mong Saetern (UTL), Stanford EH&S Biosafety and Biosecurity Office.

Postdoctoral and PhD mentors:

From Gao and Dhanasekaran Labs, Santiago Mille Fragoso, Dr. Dawiyat Moussadi