SAFETY

iGEM Policy

All of our parts and organisms are compliant with the iGEM White List.

Organisms

  • Phaeodactylum tricornutum SAG 1090-1a obtained from the Culture Collection of Algae at the Georg-August University of Göttingen
  • Pseudomonas fluorescens MTCC 103 from the Microbial Type Culture Collection, Department of Biotechnology, Government of India

Parts / BioBricks

  • nhoA (arylamine N-acetyltransferase) from E. coli, synthesized by Twist Biosciences
  • 4abh (anthranilate to 4-aminophenol) from Agaricus bisporus, synthesized by Twist Biosciences
  • 4(S)-limonene synthase from the iGEM Distribution Kit 2024
  • J23100 constitutive promoter from the iGEM Distribution Kit 2024
  • B0031_m1 RBS from the iGEM Distribution Kit 2024
  • B0015 terminator from the iGEM Distribution Kit 2024

Project Safety

Containment and Isolation

In establishing our proof of concept for In Situ Resource Utilization (ISRU) on Mars, maintaining rigorous safety protocols is paramount to ensure both the integrity of our research and the protection of the environment and personnel involved. At every stage of the project, from the design of our synthetic biology systems to the experimentation phase, we are adhering to biosafety and biosecurity guidelines to mitigate any potential risks associated with the organisms and compounds we are engineering.

Containment and Isolation

All genetic modifications of Phaeodactylum tricornutum and Pseudomonas fluorescens are conducted under strict containment in a biosafety level 1 (BSL-1) laboratory, as both organisms are classified as low-risk and non-pathogenic. Cultures are grown in sealed containers, and all experimental procedures involving live organisms take place within biosafety cabinets to prevent unintended release. For Martian soil simulants, we ensure that materials are sterilized prior to and after use, eliminating any potential cross-contamination.

Genetic Engineering

For genetic modifications, we are utilizing Golden Gate Assembly methods under stringent guidelines to avoid off-target effects and ensure the stable integration of desired pathways. All plasmids and genetic parts used in the project were carefully sourced through the iGEM Registry or a company (Twist Biosciences) following the International Gene Synthesis Consortium (IGSC) guidelines to avoid the synthesis of harmful or dual-use sequences. Additionally, we are employing well-characterized bio-bricks to reduce the risk of unforeseen interactions between synthetic and native metabolic pathways.

Chemical and Biological Hazard Management

The use of engineered bacteria to produce limonene and diatoms to synthesize acetaminophen involves handling potentially hazardous chemicals. All work with organic solvents, such as those used for the extraction of limonene, takes place under a fume hood to prevent exposure to harmful vapors. Additionally, safety data sheets (SDS) for all reagents, including acetaminophen precursors and solvents, are readily available, and lab personnel are trained in their proper handling and disposal. Through our lab work, we will be using carcinogens, mutagens, highly flammable chemicals and acid and corrosive chemicals, through ethidium bromide, ethanol, and organic solvents for limonene extraction.

Environmental Safety

We appreciate the danger of the potential environmental hazards posed by waste generated during the project. To address this, all waste products, including diatom cell debris and bacterial cultures, are treated with chemical disinfectants or autoclaved before disposal, ensuring complete neutralization of any active biological material. Wastewater from the experiments is treated with bleach to remove any residual microorganisms.

Risk Assessment and Incident Response

Prior to commencing the project, we conducted a thorough risk assessment, evaluating the potential hazards associated with each step of the process. Standard operating procedures (SOPs) have been developed to cover everything from the handling of genetically modified organisms (GMOs) to emergency response protocols in the event of accidental release or exposure. All team members are trained in these SOPs and regularly review safety measures to stay prepared for unexpected situations.

Lab members are required to wear PPE at all times when handling the fine particulate Martian soil simulant to prevent lung or eye damage, including gloves, lab coats, and eye protection. Additionally, specialized gloves are used when working with solvents or potentially hazardous materials. All protective equipment is disposed of after use to avoid cross-contamination.

Mitigating Future Risks

Our future biocontainment strategy involves the integration of a dual kill switch system combining a tetracycline repressor (TetR)-based mechanism and the ReIE-ReIBE toxin-antitoxin system to ensure robust containment of engineered organisms. See more about this system here.

Institutional and Lab Safety

At our institute, IIT Madras, lab safety is a top priority, and we collaborate closely with lab managers and lecturers who have considerable expertise controlling hazards in lab settings. Our team structure requires us to disclose any dangers or risks to a supervising panel of professors. They then determine if the activity requires graduate student supervision as well as additional assistance for a specific assignment.

Because of the complexities of the diatom-bacterial co-culture system and the possible hazards of dealing with genetically altered organisms, we sought extra help from iGEM's Safety Committee, bioethics experts, and institutional biosafety officers (IBOs) to assure the project's safety. All members of the lab team underwent extensive safety training on issues such as lab access regulations, biosafety level differences, right use of biosafety equipment, good microbiological tactics, sterilization and disinfection protocols, and emergency response procedures. Our safety instruction included dual-use research considerations, as well as chemical, fire, and electrical safety.

We meticulously followed laboratory safety regulations, which included wearing personal protective equipment (PPE), reporting incidents, maintaining inventory, and ensuring physical biosecurity. We kept detailed records of lab access and data handling, performed proper waste management, and used containment methods to avoid unintentional exposure to our modified diatoms and bacteria.

When working with our modified bacteria, we followed the advice of graduate students who had substantial expertise handling these organisms, ensuring that all best procedures were followed. Extensive conversations with lab personnel and safety officials confirmed that all lab processes met the essential safety requirements. We also created explicit procedures for our staff, identifying possible dangers and the necessary safety steps to mitigate them.

Throughout, we have maintained constant communication to ensure that all lab work is completed in a safe, controlled atmosphere. Due to our undergraduate status, we made certain that everyone received adequate training and supervision from experienced graduate students, while adhering to defined risk-mitigation measures.

Professors, graduate students, and laboratory personnel constantly maintain and regulate access to our lab. These efforts ensure that the lab work is done safely and that all team members are provided with the knowledge to operate responsibly, and so reducing uncertainties and risks throughout the project.

Our Lab