SAFETY

Safety, being an integral part of any research project, helps in analyzing the feasibility of any work. It gives the researcher an insight into the probable ways their work could be harmful to humans or the environment as a whole.

For iGEM 2021, we focus on engineering recombinant chitinases with enhanced antifungal activity than its wild-type. Chitinases, being hydrolytic enzymes, if accidentally released into the environment, can cause potential harm to non-target organisms like insects.

To ensure the overall safety of the project as well as our fellow team members in these pandemic times, several measures have been taken and followed religiously.

Lab Safety:

1. Personal protective equipment:
   a. Lab coats must be worn while working in labs and not outside the lab.
   b Gloves, especially while handling toxic chemicals and carcinogenic substances.
   c. Safety goggles must be worn to avoid any spills in the eyes.
   d. Face masks, especially with minute pore sizes should be worn. This prevents any inhalation of fungal spores.
2. Washing the hands with 70% ethanol before and after use of the lab.
3. No food or drinks in the lab.
4. Each member of the team should familiarize themselves with the location and use of safety showers, fire extinguishers, eyewash stations, and fire alarms.
5. Several short notes are stuck at various locations in the lab, indicating some lab rules, reminders of any specifications of any apparatus to be used, etc. This helps in keeping a check that one does not forget to follow the code of conduct in a lab.

Chemical Safety:

Several hazardous chemicals are being used by the team. To ensure safe handling and proper disposal of the chemicals, certain measures must be taken: MSDS of the chemicals is read and noted of any serious dangers.

1. For EtBr and Beta-mercaptoethanol, the gloves and tips used are immediately discarded in the yellow bin. A dedicated set of pipettes was designated for handling these chemicals which were cleaned with ethanol subsequent to each use.
2. The agarose gel containing EtBr is discarded in the yellow bin.
3. The corrosive chemicals are handled in a chemical hood wearing gloves, covered shoes, lab coats, masks, and goggles. This prevents any injury in case of any spills.
4. Any cytotoxic waste such as TEMED (used in the gel for SDS-PAGE) is discarded in the yellow bin.
5. Biomedical Waste segregation guidelines:
   a.Red Bucket: Gloves, microtips and microtubes, syringe without needles, culture plates without media, pipettes, plasticware
   b.Yellow Bucket: Face mask and caps, cytotoxic waste, agarose gel (containing EtBr), acrylamide gel (PAGE), pathological waste, solid culture media, body fluid/cytotoxic contaminated paper, cotton, swabs, and cloth, tissue and wipes contaminated with cytotoxic waste
   c.White Translucent Bucket-Sharps: Needles, syringes with fixed needles, blades, scalpels
   d.Blue bag (Glasses): Broken Glass, ampules, microscopy slides
   e. Blue bag (Metals): Metallic nails, metallic parts and scissors
6. General Waste segregation guidelines:
   a. Blue Bin:Paper- uncontaminated, tissue and wipes-uncontaminated, plastics- foils, and wraps including serological pipette cover, cardboard- glove box, reagent kit package, plastics from packed snacks/food, other non-toxic, non-hazardous waste
   b. Aluminum bin: Aluminium foils and wraps
7. Fungal waste disposal:
   a. After transfer work was completed, the area was cleaned again with a 70% ethanol solution.
   b. Hands were cleaned with 70% ethanol and subsequently with soap and water.
   c. The used plates were covered in plastic cover(polypropylene) and kept in an autoclave. After completion, they were sent out for incineration. (If it was not possible to autoclave old stock cultures and glassware, they were left overnight with 70% ethanol or bleach, after which they were incinerated.)
   d. We have a separate autoclave machine for decontamination purposes. The autoclave is then thoroughly cleaned after decontamination of fungal waste.
   e. The autoclaved glasswares were kept in the chromic acid solution overnight and taken out wearing gloves, followed by washing them with lab-grade soap and drying the glassware in a hot air oven for reuse.
   f. Any other waste produced was segregated and disposed of accordingly.
   
8. Working in a lab in a safe environment needs proper training. Moreover, we have also been working with fungus, isolated from the environment.
   a. The team members, especially people carrying out the experiments have been mentored by lab assistants and Ph.D. students. They have trained the members to use various apparatus, helped with protocols, guided in hands-on experience with experiments in cloning and purification of protein.
   b. Dr. Mohammed Aiyaz, the advisor of the team is a trained mycologist and had trained some of the team members to work with fungal samples. He had guided us through the collection of the fungal samples, their identification through Sanger-Sequencing, and math models based on fungus.

Project Safety:

Chitinases are hydrolytic enzymes, capable of breaking down chitin polymers to produce monomers or oligo polymers of N-Glucosamine. Even though chitinases are not a potential hazard for human health, they can potentially harm non-target organisms in the environment such as insects or any other organism bearing chitin polymers on its exoskeleton. To ensure the safety of the environment from our protein of interest and to contain our samples to the lab settings only, certain measures have been taken such as:

1. The chassis used in our project includes E.coli strains DH5-Alpha and BL21(DE3). These strains do not carry the well-recognized pathogenic mechanisms possessed by E.coli strains causing enteric infections, making them highly unlikely to cause any pathogenesis. (reference)
   
   Additionally, the DH5-Alpha strain lacks the gene recA to increase insert stability.
(reference)
2. The gene is expressed in E.coli BL21(DE3) which will be under the control of an IPTG inducible promoter. Thus the expression of the protein is possible only in presence of the inducer, which will mitigate the risk of accidental leakage.
3. We found out from literature surveys that the chitinases that we selected, when expressed by E.coli get trapped in inclusion bodies. This is further supported by the SDS results of our recombinant chitinase which shows that the entire fraction of our recombinant protein remains in the inclusion bodies and cytoplasm. This further prevents mass exposure of our enzyme into the environment.
4. The planned experiments are done in the BSL1 facility equipped with the following:
   a. Biosafety cabinets (class II type A2) equipped with a HEPA filter.
   b. Laminar airflow cabinets
   c. Autoclave machine for decontamination purposes
   d. Incubator-shaker
   e. Chemical fume hood
   f. Colored bins for proper and safe segregation of wastes
5. Experiments are conducted after seeking due guidance and training from our PI, advisors, and Ph.D. mentors. The data or results obtained are analyzed, interpreted, and presented to them for their guidance and conclusions.
6. There will not be any deliberate release of the transformed E.coli outside its growth medium.
7. The used plates and cultures are disinfected using a bleach solution, autoclaved, and sent for disposal according to the safety norms of the institute.
8. The recombinant proteins, given successful purification, will be used in a controlled manner for checking their chitinase activity following all standard protocols and lab safety norms.
9. Moreover, it has been taken utmost care to use organisms belonging to only risk groups 1 and 2. Upon identifying fungus, we looked up their risk group in various databases. The fungal species with no reliable source of risk group were further not considered for experimentation. F.solani has been marked under risk group 3 in Regulations and Guidelines for Recombinant DNA Research and Biocontainment 2017, DBT, India. The new Draft List of Microorganisms Corresponding to different Risk Groups classifies it under risk group 2, but the draft is not under effect yet. Hence, owing to this ambiguity, F.solani was also not considered further for experimentation.
10. Our project has been appproved by the Instituional Biosaety Committee(IBSC).

Human Subject Research Safety:

The team has conducted surveys and held webinars for better outreach of the project. Since this involves Humans Subject Research and reaching out to people, certain precautions are taken into consideration:

1. The survey released by the Human Practice team had been approved by the Institution before sending out to people.
2. The survey necessarily mentioned that the form can be filled by adults themselves. If being a minor, they would need prior consent from their guardian to take part in the survey.
3. Participant consent was sought in the initial of the form, and it was expressly communicated that the information collected will be solely used for project purposes.
4. For the webinars conducted by the team, consent was sought from the registered participants for using the recordings of the webinars in social media channels (Youtube, etc).

2021 still hasn’t seen the dawn of the covid-19 free world, yet being a responsible citizen, we ought to follow certain guidelines to ensure that everyone in the team and the campus are safe.

Certain guidelines have been imposed by the institute to ensure this:

1. Face masks must be worn at all times on campus (except while playing and while eating/drinking). It should be worn properly covering both mouth and nostrils.
2. We should maintain a safe distance of 6ft (2m) from each other at all times, not move around/gather in groups of not more than 3 on campus. No more than 4 people should be in any of the stores on campus at one time.
3. Mess timings have been staggered to allow only a minimum number of students at one time in the CDH (Central dining hall). We should not spend more than 30 minutes at CDH for each meal session.
4. Maintaining proper distance while standing in the queue and avoiding crowding at the counters at the cafes and CDH. No more than 3 people are allowed to occupy each dining table at one time.
5. Washing hands with soap or hand sanitizers as frequently as possible. If we touch a surface, which could be contaminated, we must sanitize our hands immediately. Before entering the CDH area, we must sanitize our hands.
6. We should avoid using lifts, not more than 2-3 persons should enter the passenger lift at one time and not touch the hand railings while using the stairs.
7. We should not visit other rooms in the hostel or use toilets/bathrooms on other floors, even at the lab area.
8. We should stay in the hostel room if feeling unwell or have any COVID-19 like symptoms, seeking advice on health conditions from the health center is recommended.