Laboratory safety is a primary condition for all our experiments. Our project involves no pathogenic organisms, no biological toxins and no non-standard lab reagents. Our experimental designs were vetted by our Ph.D. mentors, who also trained us in lab safety and sterilisation practices. Our team is in full compliance with the safety and security policies of the iGEM competition. The Department of Biotechnology[1,2] India also oversees the implementation of biosafety guidelines in biological research labs across the country and these are strictly adhered to by our institute. Provided below is a list of safety practices we followed throughout the project.
Standard Lab Safety
- Laminar flow hoods were put under UV radiation for twenty minutes multiple times a day.
- The hood was always sterilised with a 70% ethanol solution.
- All reagent vessels introduced into the hood - eg: small tubes of antibiotics - were placed in one corner of the front of the hood and aliquots were taken from there via a sterilised micropipette. If a reagent was not radiation-sterilised/autoclaved, it was sterilised using a syringe and a 0.22 micron filter.
- Before any sterile liquid/culture was transferred from one vessel to another, the mouths of the vessels were sterilised using a flame from a burner in the flow hood.
- No food or drink was allowed in proximity to the sterile spaces in the lab.
- Towards the end of our project, there was concern about a potential phage outbreak in some labs on campus (E. coli cells were mysteriously undergoing lysis). This led to a massive sterilisation drive, including in our lab. The laminar flow hood was UV-sterilised overnight, and the incubator was dismantled and washed with isopropanol and ethanol. We took the decision to ethanol-wash and UV-sterilise all autoclaved flasks before use, just in case.
Microorganisms and Parts
- The organisms we handled - the butanol-producing E. coli KJK01 and S. elongatus UTEX 2973 - are both of biosafety level one. Standard laminar flow hoods and sterilisation practices were sufficient for their containment.
- All parts expressed in these strains - cscA, cscB, cscK, atoB, hbd, crt, adhE2, sps, spp and ter - have no hazards associated with them, and all originate from BSL1, non-pathogenic organisms(including E. coli W, Clostridium acetobutylicum, Treponema denticola and Synechocystis PCC 6803. We have not isolated any new parts, nor strayed outside the whitelist.
- All cultures were hypochlorite-killed before disposal.
- All strains were sent to us as cryopreserved stocks or plates in sealed packaging.
Our Project Design
- Our project did not involve release of either organism outside the laboratory. All experiments took place inside sterile environments, and all plates and supernatants were stored below 4°C
- Antibiotics were used to maintain pure cultures of the engineered strains. If a culture had to be grown without antibiotics as the control strain (WT) lacked antibiotic resistance, a pure culture was maintained overnight in antibiotics, and then passaged into an antibiotic-free medium.
- Strong corrosive acids and bases - KOH and HCl - were handled by team members wearing nitrile gloves.
- Mutagens and carcinogens like ethidium bromide and phenol chloroform were likewise handled using gloves.
- Guidelines and Handbook for Institutional Biosafety Committes (IBSCs), 2011 https://dbtindia.gov.in/sites/default/files/uploadfiles/Guidelines%20_Handbook_2011.pdf
- Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017
Regulations and Guidelines.pdf