Team:IISER-Pune-India/Safety
Laboratory Safety
Our team is in full compliance with the safety and security policies of the iGEM competition. The Department of Biotechnology, India also oversees the implementation of biosafety guidelines in biological research labs across the country and these are strictly adhered to by our institute. Each member was required to undergo mandatory training sessions about safe lab practices and biosafety from our Ph.D. mentors and Dr. Sanjeev Galande (PI), who have experience working in the lab and have the necessary knowledge in general lab safety and precautions to follow. They have experience working in the laboratory setting and are well informed about general lab safety and precautions that need to be taken in the lab to oversee and guide our wet lab members as they perform the experiments. The safety training was completed through online means. The few wet lab members who were able to gain access to the labs were given separate mandatory training in the lab.
Safety in experiments
The organisms that our team has used are specially engineered to survive only in optimum laboratory conditions. These strains do not pose much of a threat to lab personnel or anyone inside or outside the lab given their inability to survive outside lab conditions. We have used E.coli DH5α for the cloning process and E.coli BL21 DE3 origami and E.coli BL21 AI as the chassis for producing the grafted cyclotides and the parasite and human proteins. These organisms are classified as risk group 1 organisms and are permitted to be used as biological vectors. International guidelines on biological safety declares them as prototypes of biologically safe vehicles and as a result pose no risk to lab personnel. We have ensured the safe disposal of any bacterial waste, for instance, by treating glassware with bleach and nucleases to digest any plasmids used in the experiments.
The Plasmodium parasites which were to be used for some of the experiments are grown in an enriched RBC medium and are unable to survive outside this enriched environment. This means that these parasites are not particularly dangerous to the members of the lab, or to the outside community. Due to the limited time we had in the lab, we were not able to work with the Plasmodium parasites. However, certain safety precautions would have been taken if we were to work with Plasmodium parasites.
Due to the COVID-19 pandemic, we were unable to gain access to the lab until recently which meant we were not able to perform most of the experiments that we had planned. Most of our work was completed through online means. However, we have considered the potential biosafety concerns for all our experiments that are highlighted in the experiments section.
Project Safety
The native cyclotide backbone is known to have hemolytic activity which could be a hurdle when used as a drug delivery system. Through literature, we have identified that loop1 and loop3 of the cyclotide KalataB1 contribute most to the hemolytic activity of the cyclotide with loop3 being the most prominent. With the aim of reducing the hemolytic activity of the cyclotide we replaced the amino acids in loop 3 with a strep-tag. This in turn will help in the identification of the inhibitory peptide. From the results obtained from the literature, we know which residues of loop 1 and loop 3 contribute to the hemolytic activity of the cyclotide KalataB1.[1] We aim to mutate those specific amino acids and hope that these mutations will reduce the hemolytic activity of the cyclotide. The complete details of the mutation assay are mentioned in our experiment section.
Due to limited lab access, we were not able to validate the reduction in hemolytic activity of the cyclotide after adding strep-tag in loop 3 and were not able to perform mutations in loop 1 of the cyclotide Kalata B1.
References
[1]. Simonsen SM, Sando L, Rosengren KJ, Wang CK, Colgrave ML, Daly NL, Craik DJ. Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity. J Biol Chem. 2008 Apr 11;283(15):9805-13. doi: 10.1074/jbc.M709303200. Epub 2008 Feb 7. PMID: 18258598.