Overview
Our project aims to achieve the de novo biosynthesis of ginsenoside Ro in an engineered Saccharomyces cerevisiae W303 strain using glycerol and fatty acids as carbon sources. The core of the work involves standard molecular biology techniques—constructing plasmids and transforming them into a Biosafety Level 1 (BSL-1) strain. We provide a comprehensive safety assessment covering laboratory practices, the safety of the engineered strain, and the safety considerations of the final product.
Laboratory safety
→ Before starting any wet-lab work, all team members received mandatory safety training. The training covered:
·Standard microbiological practices: no eating or drinking, use of personal protective equipment (PPE), handwashing, etc.;
·Proper handling and disposal of biological materials: autoclaving, waste management, etc.;
·Chemical safety: precautions for handling hazardous chemicals and emergency response procedures for accidents.
→ Our work is confined to BSL-1 facilities, and the primary hazards arise from standard laboratory procedures rather than the biological agents themselves:
· Electrical safety: use of gel electrophoresis equipment, water baths, and centrifuges;
· Chemical safety: common HPLC mobile phases (acetonitrile, methanol, etc.) are flammable/toxic—prepare and collect waste in a fume hood; handle ethidium bromide for gel staining in a designated area while wearing gloves;
· Personal safety: use of open flames and UV transilluminators;
· Waste disposal: all biological waste is autoclaved for inactivation prior to disposal; chemical waste and sharps are segregated and handled according to university regulations.
Project safety
A. Chassis organism
We use Saccharomyces cerevisiae W303. It is a well-studied, non-pathogenic Risk Group 1 (RG1) organism that cannot survive in the human gut or in natural environments outside the lab, and it poses no known threat to human health or the environment.
B. Multi-layer biological containment
· Auxotrophic markers: The engineered yeast strains are auxotrophic for specific amino acids (e.g., tryptophan, leucine). They can only survive on laboratory media supplemented with these nutrients and cannot grow in nutrient-limited natural settings.
· Plasmid system: Key metabolic functions are plasmid-encoded. Once outside the selective pressure of laboratory media, the plasmids are likely to be lost over passages, reverting the yeast toward an unengineered state.
C. Environmental and community safety
Because of the above containment measures, the risk that our engineered yeast survives outside the laboratory or transfers engineered genes to other organisms is extremely low. Even in the highly unlikely event of accidental release, auxotrophic yeast would be unable to propagate.
D. Chemicals
The table below lists the hazardous chemicals our team used during the iGEM competition, along with their associated risks and protective measures.
| Chemicals | Related risks | Protective measures |
|---|---|---|
| NaOH | Highly corrosive, causes burns to skin/eyes | Goggles, face shield, alkali-resistant gloves; preparation and transfer with splatter protection |
| Chloroform | Causes skin irritation and serious eye irritation, harmful if swallowed,
toxic if inhaled, may cause drowsiness or dizziness, suspected of causing cancer |
|
| Sodium orthovanadate | Toxic; harmful to the liver/blood and other systems | Fume hood, gloves; avoid contact with dust/solution |
| NaF | Toxic, ingestion/inhalation can cause poisoning | Gloves and goggles; avoid accidental ingestion |
| Glacial acetic acid | Strongly corrosive, vapor irritant; incompatible with alkalis/oxidizers | Fume hood; acid-resistant gloves, goggles |
| Ethidium bromide | Mutagenic; harmful to skin/eyes/environment | Wear chemical-resistant gloves and safety goggles; use specialized
laboratory equipment,
and collect hazardous chemical waste separately for proper disposal. |
| Ethanol | Highly flammable, causes skin irritation, and serious eye damage | Keep away from fire sources; seal containers; use a fume hood if necessary |
| SDS | Causes severe irritation to the eyes and skin Inhalation of the powder may irritate the respiratory tract |
Wear a mask when weighing to prevent inhalation, and wear gloves and goggles |
| APS | Strong oxidizer; skin/respiratory sensitizer | Keep away from flammable/reducing agents; wear a protective mask |
| TEMED | Corrosive/Irritant; Flammable | Ensure good ventilation; avoid contact with skin and eyes |
| DMSO | Has extremely strong permeability and can easily penetrate the skin and most gloves | Solvent-resistant gloves |
| LiAc / LiOAc-1% SDS | Irritating to eyes, skin, and respiratory system; harmful if ingested | Gloves, goggles; avoid aerosols |
| Coomassie Brilliant Blue R-250 staining solution | The dye itself is irritating; the formulation contains methanol/acetic acid → flammable/corrosive | The entire process of staining/decolorization is carried out in a fume hood; in a sealed container |
| Tween-80 | May cause mild irritation to the eyes and skin | Wear gloves |
| Chloroform/methanol mixture | It combines the toxicity of chloroform with the flammability of methanol, resulting in compounded risks | Fume hood, away from heat sources and open flames |
| Ethyl acetate | Highly flammable, irritating to eyes and respiratory system | Keep away from heat sources and open flames, fume hood,solvent-resistant gloves and safety goggles |
| Methanol | Flammable liquid. Vapors can form explosive mixtures with air. It has
specific toxicity
to the visual and central nervous systems, which can cause blindness or even death |
Keep away from heat sources and open flames, fume hood, solvent-resistant gloves and safety goggles. Avoid inhalation and skin contact |
| Acetonitrile | Flammable liquid. Vapors can form explosive mixtures with air.
Inhalation, ingestion, or
skin absorption may cause poisoning, with symptoms including fatigue, nausea, and respiratory depression |
Keep away from heat sources and open flames, fume hood, solvent-resistant gloves and safety goggles |
| Acrylamide | Has strong neurotoxicity, can be absorbed through the skin, and is a suspected carcinogen | Chemical-resistant gloves and safety goggles |
Product safety
Our target product is ginsenoside Ro, a natural compound found in multiple Panax species with a long history of safe use in traditional medicine and consumer products. The compound itself is not hazardous.
· Production and purification: Ginsenoside Ro is produced intracellularly in our engineered yeast. Only highly purified Ro small molecules are output—containing no viable GMOs or replicable biological material. Multi-step purification (extraction/chromatography/HPLC) with solvent recovery ensures residual solvents and host DNA/proteins remain below regulatory limits.
· Future applications: If this production method is scaled, the purified ginsenoside Ro—whether intended for cosmetics, functional foods, or pharmaceuticals—will be subject to all relevant safety regulations and oversight by competent authorities (e.g., National Medical Products Administration).