Biosafety is the safe working practices associated with the handling of biological materials. It addresses the containment principles, methods, and procedures employed to prevent unintended exposure to diseases and poisons, as well as their inadvertent release. Responsible laboratory methods, including security, management, and accountability for critical biological materials, will aid in preventing unauthorised access and misappropriation of work. For iGEM 2022, our project aims to develop a bacterial nanovesicle-based drug delivery system for use in breast cancer. Several precautions have been adopted and are being strictly followed to guarantee the general safety of the project as well as of our fellow team members.
The safety of the project design is made up of the safety factors that led to the creation of our drug delivery system. This includes reducing off-site targeting, reducing LPS reactogenicity, and selecting effective prodrug-enzyme systems.
Handling of the bacterial cultures is done inside LAF and wearing protective equipment. Since the isolation of OMVs requires the use of high-speed centrifuges, only a trained team member is allowed to use these machines. Specific protocols are in place in case of some property/equipment damage. Decontamination protocol is followed at the end of the isolation, which involves proper waste segregation, cleaning the equipment used, and autoclaving the bottles/tubes used. We have an approved check-in form for our attenuated fusion protein.
He is an Associate Professor in the Chemical Engineering department at the Indian Institute of Science, IISc and also an Associate Faculty at the Centre for BioSystems Science and Engineering.
We met with Dr. Rahul to gain insights into how to safely engineer Cytolysin A as a display system for our OMVs. He clarified that the protomeric form of ClyA was the predominant conformation in the bacterial outer membrane and that ClyA does not cause significant leakage of cargo from vesicles or of periplasm from bacteria, which strengthened our choice to use ClyA in our project.
We also learnt that specific substitution mutations could be made to attenuate ClyA, and it could effectively prevent its toxicity by inhibiting oligomerisation.
This mutation helped us secure the required level of safety for our Cytolysin A study.
Figure 2. Left- Dr. Rahul Roy, Right- Meeting with Dr. Rahul Roy
To ensure that attenuated ClyA does not cause cytotoxicity and cell death, we performed a MTT cell viability assay using OMVs containing ClyA-3xFLAGtag-THP against CX3CR1 on SK-BR-3 cells. We can conclude that the bacterial strain expressing THP against CX3CR1 would give us OMVs, which would inherently have very minimal or no cytotoxic effect on mammalian cells. The MTT assay result is discussed in detail under the results section.
We acquired our test cell line - SK-BR-3, from Dr. Annapoorni Rangarajan, Assistant Professor, IISC-Bangalore and from Dr. Santhosh Kumar T.R, RGCB. Cell culture experiments are done inside the biosafety level-1 cabinets with utmost sterile conditions. UV sterilisation is practised before and after the experiments are done. Plates/Equipment/Materials which came in contact with cultures are washed with 5% bleach solution followed by ethanol wash before being discarded. Only trained team members are allowed to handle cell culture experiments to avoid spillage and accidental exposure.
Our project has been approved by the IBSC (Institutional Biosafety Committee). Our research and work development applications were considered and noted by the Review Committee on Genetic Manipulation (RCGM under DBT) in its 228th meeting held on March 17, 2022. Our PhD mentors and advisors guide us at each stage of the project, and the institute safety officer inspects and approves all aspects of our proposal.
Safety awareness is a constant realisation that people must have at all times. It extends beyond what we hear in safety seminars and the classroom. In order to reduce the risks associated with safety, it is essential to be continually aware of how things work and capable of identifying and foreseeing hazards. In other words, we can say that "Awareness is Safety". So we decided to extend safety awareness beyond what's usually done and introduced: Track your Waste and Crime Scene Investigation (CSI).
Track your waste was a team initiative in which we tracked the journey of waste generated in the lab from generation to the final management step. We did intensive research and a series of interviews with staff handling the waste, people involved in the transportation of the waste to the treatment plant, and experts at the plant. We summarised all the information we learned into a documentary and shared it on social media platforms as a humble initiative for spreading awareness and maintaining continual awareness. You can find the pdf document detailing all of our findings throughout the course of our journey following the waste produced by our institute in the HP Section and the documentary film on the Education page.
Documentary film screening in the Seminar Hall
Crime Scene Investigation, a mystery-filled online game meant to help university students learn about biosafety and the rules and regulations that govern bioethics. The aim of the game is to predict a plausible explanation for the crime in the game, and the theme for our game was based on the importance of biosafety. We were successful in conveying our message through a divergent approach.
As decided by our team, we ensured that all the members attended thorough safety and security training, including handling bacterial cultures and disposal of contaminated materials before being allowed into the lab. Our institute’s biosafety officer Dr Poonam Thakur mentored us during the safety and security workshop, which covered topics of:
Image from Biosafety seminar by Dr. Poonam Thakur
We scrupulously followed the general safety requirements, as well as the biology department safety guidelines. iGEM safety rules and policies were also followed while doing research activity in our lab.
Images of the lab workspace.
Chemical safety is achieved when all activities involving chemicals are done in a way that protects human health and the environment. Extraction or synthesis, industrial production, transport, use, and disposal of all substances, both naturally occurring and manmade, are included, as well as the full spectrum of exposure situations. Chemical safety has many scientific and technical parts.
During the span of our project, the team conducted several online and offline meetings, webinars, surveys, interviews, documentaries, camps and competitions to spread awareness and also to gain insights for improving our project. Since this involves Humans Subject Research and reaching out to people, we made sure to take all relevant precautions and abide by relevant laws and regulations, as well as institutional rules or guidance.
For both the hair donation and breast cancer screening camp we had got signed a memorandum from the person or organisation we collaborated with to ensure that both parties adhered to their responsibilities.
We also got written consent from the participants mentioning they were informed about all the camp details and that they willingly participate in the program.
