Our team has always prioritized the well-being of the laboratory, team members, and the environment. All experiments have been conducted in a manner that is appropriate to institutional and national guidelines. This ensures proper handling, storage, and disposal of equipment and materials. Our primary investigator has guided our work at every step, allowing us to be accountable and maintain transparency.
Identifying Project Risks
The team consulted experts from research institutions and government organizations to understand the levels of risk to be encountered through our work. These discussions allowed us to conclude that the risks are minimal in themselves, as long as the appropriate biosafety measures are maintained. The cell-free nature of our solution avoids risks associated with a chassis/in vivo system. Off-target effects with respect to the siRNA are mitigated through the stringent design and validation process.
Handling these Risks
Our Laboratory
All experiments are conducted in the Biotechnology department's Project Laboratory, which is designated as Biosafety Level 2 (BSL-2), in accordance with the guidelines outlined by the Indian government and the Centre of Bioethics at Manipal Academy of Higher Education (MAHE). A Class-2 Biosafety cabinet with an ULPA filter is used to ensure the safety of microbial cultures, chemicals, and the environment. UV treatment is done prior to every use of the cabinet.
Chemical fume hoods, emergency eyewash stations, and full-body showers are some of the facilities present to ensure the safety of working members. Sterilization of working surfaces is done using 70% ethanol. All laboratory waste is segregated into colored bins that are promptly disposed of by lab technicians.
Equipment in the lab, such as centrifuges, autoclaves, and storage units, is regularly serviced. The laboratory is fumigated every 3 weeks to maintain sterile conditions.
Laboratory and Safety Training
Our wet lab members have received extensive training in standard molecular biology techniques such as PCR, bacterial transformation, and SDS-PAGE. In addition to technical skills, we have also undergone training in the safe and proper use of essential lab equipment such as the autoclave, biosafety cabinet, centrifuge, etc.
The training has allowed us to develop an understanding of risk assessment protocols, including the identification of biological, chemical, and physical hazards. We understand the importance of following standard operating procedures (SOPs) and using appropriate Personal Protective Equipment (PPE) such as lab coats, gloves, masks, and safety goggles during experiments. All biohazardous waste is decontaminated before disposal. Chemical, biological, and material wastes are separated and disposed of in accordance with the institutional safety guidelines.
Regulatory Compliance
The following safety regulations have been reviewed to ensure compliance:
- The Environment (Protection) Act, 1986: Provides guidelines for the disposal of synthetic biology-related products and handling of hazardous substances (Parliament of India, 1986).
- Rules for the Manufacture, Use/Import/Export, and Storage of Hazardous Micro-Organisms/Genetically Engineered Organisms or Cells: Oversees safe practices in experimentation and waste disposal (Ministry of Environment & Forests, 1989).
Hazardous Chemicals
The following are chemicals used in our experimentation that pose a safety risk. Utmost care is taken while performing any procedure using these chemicals.
Highly flammable chemicals:
- Ethanol: A highly flammable solvent used for sterilization purposes. It is stored away from fire hazards.
- Isopropanol: Highly flammable and serves as a sterilizing agent. It is stored away from fire hazards.
- Methylene blue: Produces hazardous combustion products like Nitrogen oxides (NOx), Carbon monoxide (CO), Sulphur oxides (SOx). Used to stain P. capsici zoospores. Direct exposure and contact with the skin are avoided.
Acids and corrosive chemicals:
- Hydrochloric acid: An acid capable of causing severe skin burns on contact. It is used for buffer preparation and pH adjustment. Direct skin exposure is avoided.
- Sodium Hydroxide: A base capable of causing skin burns and irritation. It is used to adjust the pH of our nanoparticle solutions.
- Acetic acid: A corrosive acid that can cause burns. 1% acetic acid is used to dissolve chitosan in the preparation of nanoparticles. Acetic acid is also used as a control in the detached leaf assay.
- Lactophenol cotton blue: A corrosive chemical that is a chronic hazard to aquatic environments. It is used to visualize P. capsici under the microscope.
Other hazardous chemicals:
Diethyl Pyrocarbonate (DEPC): An irritant and carcinogen used to inactivate RNases. It is used to treat all apparatus that comes in contact with the siRNA.
Phytophthora capsici
P. capsici is a Risk Group 1 pathogen. Although not on the iGEM White List, a Check-in Form was submitted and approved for its usage.
The culture was obtained from the Indian Institute of Spices Research and subcultured in our lab while adhering to the safety instructions provided. To mitigate the environmental risks, all work involving the pathogen is done inside a Class-2 Biosafety Cabinet while wearing Personal Protective Equipment (PPE). Used media and glassware are autoclaved, and lab technicians ensure their proper disposal.
References
Ministry of Environment & Forests. (1989). Rules for the manufacture, use/import/export and storage of hazardous microorganisms/ genetically engineered organisms or cells. Ministry of Environment & Forests. https://ibkp.dbtindia.gov.in/DBT_Content_Test/CMS/Guidelines/20181115121526033_Rules-for-the-manufacture-use-import-export-and-storage-1989.pdf
Parliament of India. (1986). The Environment (Protection) act, 1986. In The Environment (Protection) Act, 1986 (pp. 267–270). https://www.indiacode.nic.in/bitstream/123456789/4316/1/ep_act_1986.pdf