Safety and Security
Comprehensive documentation of our safety protocols, risk assessments, and security measures for the iGEM IIT-Madras project.
Our Safety Framework
iGEM IIT-Madras is fully committed to following the national biosafety guidelines established in India. Our project design and laboratory practices are in strict compliance with the "Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017" and its subsequent updates, as set forth by the Indian government's Department of Biotechnology (DBT). Our work is further overseen by the Institutional Biosafety Committee (IBSC) at the Indian Institute of Technology Madras, ensuring all protocols meet both institutional and national standards for safety and ethical conduct.
Laboratory Safety
BSL-1 Facility - E. coli Experiments
Our wet lab work for the E.coli experiments was conducted in the BSL-1 facility of the Epigenetics Lab, located in the Department of Biotechnology at IIT Madras. Our team worked under the constant guidance and direct supervision of Prof. Arumugam Rajavelu and his dedicated PhD scholars. The lab has a certified Biosafety Level 1 (BSL-1) facility, which is suitable for the well-characterized, non-pathogenic organisms used in our project.
To ensure a safe working environment at all times, we strictly followed a set of fundamental safety protocols:
Safety Protocols
- Mandatory Training: All team members underwent comprehensive training covering the safe handling of cultures, general lab safety protocols, proper equipment handling, and emergency procedures.
- Safety Gear: Personal Protective Equipment (PPE), including lab coats, gloves, and eye protection, was worn by all members at all times within the lab.
- Containment: All work involving open cultures was performed within a Laminar Air Flow (LAF) hood to maintain a sterile environment and prevent cross-contamination.
- Supervision: All the experiments were done under supervision of at least one PhD scholar in the lab.
BSL-2 Facility - Mammalian Cell Culture
Our mammalian cell culture work was conducted in the BSL-2 facility of the Systems Biotechnology and Cellular Engineering Lab, located in the Department of Biotechnology at IIT Madras. Our team worked under the constant guidance and direct supervision of Prof. Meiyappan Lakshmanan and his dedicated PhD scholars.
Project-Specific Safety
Organisms Used
All our experiments were conducted using standard, non-pathogenic laboratory strains:
Bacterial Strains (BSL-1)
- Dam-negative E. coli, procured from New England Biolabs (NEB)
- E. coli XL1-Blue strain
Both of these strains are classified as Biosafety Level 1 (BSL-1) organisms, as they are well-characterized, are not known to cause disease in healthy humans, and are widely used for synthetic biology research.
Mammalian Cell Line
Our mammalian cell culture work was done using a non-pathogenic Chinese Hamster Ovary cell line:
- CHO-K1 cell line
Parts & Genes
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. We worked with two primary plasmids:
Custom Plasmid with DNA2P-GFP
We used a custom-synthesized plasmid containing the DNA2P promoter and a GFP (Green Fluorescent Protein) gene in a pET backbone. This construct was designed and synthesized by Twist Biosciences. The DNA2P promoter and GFP gene are standard, non-toxic genetic elements commonly used in molecular biology.
Dam-dCas9-gRNA System
We utilized the Addgene plasmid #46569 as a backbone, which already contains the gene for dCas9 and the tracer RNA. To this, we added our custom guide RNA (gRNA) and a sequence for a Dam methyltransferase. The Dam sequence was derived from a native E. coli strain, and the gRNA was designed to target a specific DNA sequence. The Dam methyltransferase was linked to dCas9 using a glycine-serine linker, a common and flexible linker that maintains protein function.
Containment & Isolation
Physical Containment
Our wet lab work required the use of various instruments and devices located both within our primary lab space and in the common lab facility. All work involving open cultures was performed within a Laminar Air Flow (LAF) hood to maintain a sterile environment and prevent cross-contamination. To ensure reliability and safety, all lab equipment, including pipettes and centrifuges, was serviced frequently to keep them in good working condition. Additionally, our labs were regularly fumigated to maintain a sterile and pathogen-free environment.
Biological Containment
All genetic modifications of Dam negative strain & XL-1 blue strain 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. 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.
Chemical Safety
Our initial lab training included detailed instructions on the safe handling and proper storage of various chemical groups. To review potential risks and safety precautions, a folder containing Safety Data Sheets (SDS) for every chemical in the lab was kept in an easily accessible location. We also made conscious choices to use less hazardous alternatives; for example, we used SYBR Safe instead of Ethidium Bromide for nucleic acid staining, thereby minimizing potential risks.
Safety Protocols
Waste Disposal
We implemented a systematic approach to waste disposal in accordance with our institutional guidelines. Biological waste was decontaminated by autoclaving before being disposed of. All chemical waste was segregated and disposed of through the appropriate institutional channels.
Emergency Procedures
To ensure a rapid and effective response to any incident, our lab is equipped with readily accessible emergency equipment. Fire extinguishers, a first-aid kit, and a wash station are all maintained in clearly marked, easily reachable locations. All team members were trained in emergency protocols for chemical spills, injuries, and fire incidents.
Risk Assessment
We conducted a thorough risk assessment to identify and mitigate potential hazards associated with our project:
Risk to the Team
Our primary risks included chemical exposure and physical harm. We mitigated these through mandatory safety training, consistent use of PPE, and strict adherence to lab protocols.
Risk to the Public and Environment
Although our project utilizes BSL-1 organisms and non-pathogenic parts, we have conducted a detailed assessment to address potential risks. We specifically considered the possibility of off-target effects of our gRNA and the stability of our engineered plasmids. The design of our system using standard lab strains and established backbones minimizes the risk of unintended spread and ensures the system functions as intended, only within the confines of our lab.
Built-in Safety Features
Further on introduction of this plasmid in the cell of interest, the effects on the organism's genome is very less as the system has a very high specificity of gRNA and cas9 in general. Further any changes made are temporary since it is based on epigenetics. In order to increase control over this system further, an inducible promoter can be used which ensures expression only when the inducer is present.
Compliance and Regulations
Our project adheres to:
- Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017 (India)
- Department of Biotechnology (DBT) guidelines
- Institutional Biosafety Committee (IBSC) oversight at IIT Madras
- International Gene Synthesis Consortium (IGSC) guidelines
- iGEM safety and security requirements