1. Prologue
The team iGEM MIT-MAHE project gives topmost priority to safety and security, focusing on safeguarding laboratory members, experimentation, and the environment. Our institute's unwavering support has enabled us to fulfil our fundamental responsibility toward biosafety. We have meticulously planned our experiments and upheld proper work ethics in the laboratory to ensure that no harm was inflicted on nature or ourselves. In this regard, we have discussed our planned experiments in detail with our principal investigator, and were performed only upon her approval. This approach ensured transparency and accountability in our efforts to maintain the highest standards of safety and security.
2. Lab Safety
1. The MIT-MAHE team's wet lab members conduct their
experiments in the Department of Biotechnology's Project Lab, a lab/workspace designated with Biosafety Level 2
(BSL-2). Our laboratory and work practices within the lab adhere to the guidelines for biosafety and biosecurity
in India as well as the guidelines provided by the Centre of Bioethics, MAHE.
2. All members of the wet lab subsystem of our team have received extensive training in safe lab practices from
our PI, Dr. Ritu Raval and our advisors before they began working in the lab.
→ We were advised on the risk assessment of the reagents in use, proper identification, management, and safe
disposal of biological and chemical hazardous waste and lab consumables as well as the necessary precautions to be
taken while working with biological materials.
→ We were also briefed on the working mechanisms of all the equipment and machinery available in the lab and
on how to appropriately and safely use them to conduct experiments. They were advised on appropriately using
equipment such as the centrifuge, UV-visible spectrophotometer, etc. as well as safe handling of the autoclave and
decontamination units.
→ We were also made familiar with the location of safety showers, eye wash, first aid kits, fire
extinguishers, as well as emergency evacuation routes.
→ We received training sessions from their advisors and PhD scholars Mr Rajesh M. Gowda and Mrs. Atheena PV
on working in the biosafety cabinet and precautions to avoid contamination.
3. Some practices all members working in the lab strictly follow include:
→ Eating and drinking are strictly prohibited in the lab.
→ All members have to wear lab coats, protective goggles, and nitrile gloves as necessary whilst working in
the lab.
→ Proper sterilisation is undertaken before working in the BSL-2 biosafety cabinet.
→ All equipment and machinery in the laboratory such as the centrifuge, spectrophotometer, cold storage,
autoclave and such, are handled with care and well-maintained. Regular maintenance work is done for them.
→ Taking further precautions, the lab is fumigated every 3-4 weeks.
Use of Harmful Reagents and Procedures:
Some experimental procedures involved using harmful chemicals and reagents, however, all additional safety
measures were taken to prevent any harm.
3. Project Specific Safety
1. As our team is working with Triclocarban and its
subsequent breakdown products, 4-chloroaniline and 3,4-dichloroaniline which are hazardous in nature and possible
carcinogens, we took extra precautions while working with them by wearing double nitrile gloves, N95 masks, safety
goggles and lab coats.
2. For decontamination and disposal of Triclocarban and other hazardous compounds, we have stored all the
equipment used, i.e., pipette tips, tissue paper as well as the solutions containing these chemicals separately to
prevent the risk of spillage and exposure to untrained individuals. This will be then sent to Re Sustainability
Limited (RE), a KKR-backed company, which is one of Asia's leading providers of comprehensive environment
management services for disposal.
3. The organisms and parts that we are working with are in Risk Group 1. They are non-pathogenic and non-spore
forming and all our work is covered by the White List, hence, we aren't conducting any activities against the
guidelines provided by the competition.
4. Pertaining to our project, physicochemical methods of degrading TCC have proved ineffective. Though the
introduction of Genetically Modified Organisms (GMOs) is considered risky, if we take appropriate measures, its
use can revolutionise bioremediation globally. Keeping safety at utmost importance, our contained bioreactor
system would be designed to ensure no interference or interaction of our bacteria with the wastewater treatment
environment. Moreover, microbial biocontainment is essential to engineer safe bioremediation strategies. The
incorporation of a kill switch (an artificial system that induces cell death) only enhances our project safety and
ensures an air-tight implementation.
5. For the hardware aspect of our project, we had collected water samples from the nearby lake and ran it through
Raman spectrometer in order to discern how the unique peaks of triclocarban would vary in the presence of
naturally occurring compounds, we made sure to abide by necessary safety protocols when dealing with natural
samples, such as using falcon tubes to collect the lake water as well as wearing gloves while handling these
samples.
6. All the equipment and glassware that we use that have come in contact with the cultures that we're working with
are routinely autoclaved (decontamination) and treated with bleach before disposal.
Meeting with Lynn Ferris, Lab Manager at UNSW, Australia: Sept 1, 2023
As a lot of our wet lab protocols involved working with toxic chemicals like triclocarban, 3,4-DCA and 4-CA, we
were concerned about the safety of our team members as well as others working in the Project Lab. To check the
suitability of our safety protocols, our Wet Lab members met Lynn Ferris, Lab Manager from UNSW, Australia. As our
lab does not have an official committee to deal with safety and security in the lab, any changes would have to
happen on a voluntary basis.
Lynn helped us realise the importance of communicating these risks to the people in the workplace and raising
awareness about the same.
Her suggestions involved:
1. The use of a fume hood with carbon filter respirators while preparing stock solutions of TCC.
2. She approved our use of gloves and safety goggles while working with these chemicals, but also added that we
use double gloves if we don't have the required glove thickness.
3. She emphasised the importance of wiping all counters and workspaces with soap and water every evening.
4. She also suggested that all our lab coats should be kept inside the lab on hooks separating them such that
they do not touch each other.
5. She also mentioned how important it was to defrost the -20 degrees fridge once in a while and also go through
the contents and discard unwanted samples.
Overall, this session with Lynn proved insightful and many of her suggestions have been incorporated by us. This meeting helped us better our lab safety and project-specific safety.
4. Safety and Security Session
To equip the second years of MIT Manipal pursuing a
B.Tech in Biotechnology with valuable laboratory safety knowledge and hands-on experience, we organised a series
of Lab Safety sessions. These sessions took place on the 14th, 18th and 19th of September, 2023, with two
half-hour sessions each day, accommodating five students per session to ensure the lab remained spacious and
manageable.
The primary objective of these sessions was to familiarise them with essential safety protocols and equipment,
equipping them with a solid foundation for the upcoming years. The sessions were structured as follows:
The students expressed their gratitude for the informative and engaging sessions, and they enthusiastically embraced the opportunity to familiarise themselves with laboratory safety protocols and equipment. These sessions not only provided them with valuable knowledge but also served as an inspiring introduction to the exciting world of scientific research within our team.
5. Dual Use
What is Dual Use?
Dual Use in the context of science describes the potential of knowledge or technologies to be used by third
parties with benevolent and malevolent intentions. (European Commission, 2018)
What is Dual Use research of concern?
Dual-use research of concern (DURC) describes research that is intended to provide a clear benefit, but which
could easily be misapplied to do harm. It usually refers to work in the life sciences, but the principles apply to
other fields including engineering and information technology. (World Health Organisation, 2020)
Why is dual-use research of concern important?
DURC poses a major concern, one such case is in March 2022, a US pharmaceutical company published a paper in
Nature Machine Intelligence on AI systems for drug discovery. They aimed to improve drug toxicity prediction but
were surprised to be invited to a security conference where they discussed the potential misuse of their models in
creating chemical and biological weapons. By refining their models to search for more toxic molecules, they could
generate 40,000 lethal molecules in under 6 hours, including VX nerve agent and new molecules predicted to be even
more powerful. The researchers admitted to being naive and untrained to think about the potential misuse of their
work. Furthermore, advancing technology and the global availability through open access to the internet, also
allows sensitive information to spread.
What can be done?
To avoid such incidents from occurring in the future, we need to make sure that the upcoming scientists are aware
of this possibility and have a clear knowledge of how their projects could have a negative effect on the world. We
started with the university level by educating the students of Biotechnology (third semester) and making them
aware of the importance of dual use.
Furthermore, iGEM is not only about starting a project from scratch. In past years, iGEM teams have produced
remarkable concepts and built impressive systems that have positively impacted the world. We maintain that
refining these exceptional ideas is not only a way to pay tribute to those projects but also a crucial step
towards creating lasting scientific change.
Introspect with our project.
Degradation of a harmful compound present in wastewater treatment plants, Triclocarban into non-toxic byproducts
is the aim of our project. We are using a novel amidase enzyme coded by the TccA gene from Ochrobactrum sp.
TCC-2, which degrades TCC to toxic chloroaniline byproducts.
The aim is to introduce Ochrobactrum
sp. TCC-2 TccA gene into a chassis which can inherently degrade the toxic chloroaniline byproducts using its
natural, existing pathways. We intend to immobilise our genetically modified bacteria onto biochar, which will be
introduced into a batch bioreactor. Ideally, the bacteria should remain immobilised on the biochar and die once
the carbon source is depleted, but in the event they do not, we plan to include a kill switch. We plan to
introduce a toxin-antitoxin kill switch to ensure its biocontainment. We also intend not to use an
antibiotic-resistance gene as a selectable marker for our modified bacteria that will be introduced into the
bioreactor.
However, a closer inspection,after we learned about dual use, revealed the potential abuse of
our project, if the plasmid is isolated from the biochar and introduced to any chassis that does not have the
ability to degrade the byproducts, it could lead to the production of chloroanilines (3,4-dichloroaniline and
4-chloroaniline) which are toxic and pose a potential threat.
Delving deeper into the issue, biochar is a rich-carbon source and will prove to be so not only for our chassis
but for a wide range of non-target organisms which could cause serious problems/epidemics due to pathogenicity.