Safety is one of the most important aspects of a project. When our project was being engineered, safety was an aspect that was prioritised in every sphere. Our project aims to detect the pathogen causing bovine mastitis in cows using a cheap, on-field test kit. On detection of pathogens, we have also developed a method of killing the target pathogen by a method that does not entail the usage of antibiotics (see design). The safety issues that pertain to both these aspects of our project have been minutely checked and taken care of to the best of our abilities.
The Department of Biotechnology (DBT) of the Government of India regulates biosafety and biosecurity in research laboratories in India. The complete set of rules and regulations regarding laboratory safety can be found here. In addition to that, IISER Kolkata has its own set of laboratory safety guidelines formulated by the Institutional Biosafety Committee (IBSC). These set of guidelines function to ensure a safe laboratory work environment, minimise the risk of accidents and also to realise the benefits of a safe work environment in eliminating the hazards to the working personnel. The team initially underwent strict lab-safety training about the dos and don'ts of lab safety under the able guidance of Mr. Suvam Mukherjee and Dr G. Lekha.
Briefing about proper handling and disposal of materials including biosafety hazards as well as developing a positive attitude toward safety in laboratory operations were taught. We were also shown where to find fire and chemical extinguishers, as well as where to find emergency showers and eyewash stations, as well as how to use them. Also, the working labs were initially fumigated to destroy any pathogens that might have been suspended in the air.
Despite the COVID-19 pandemic, we were grateful to be allowed lab access to perform the experiments that we had planned. Throughout the working period, the team diligently maintained the covid-19 guidelines:
- Only the minimum number of members required to carry out the wet-lab work were called back to college to perform the experiments. The rest of the members remained at home and performed their assigned duties and contributions in taking our project forward.
- In the lab, social distancing was maintained. The team employed two labs for carrying out the experiments and at any point of time there were no more than 6 people working in both the labs combined.
- During lab hours, the team obeyed COVID regulations by wearing the mask mandatorily, gloves and periodic sanitisation of hands and surfaces.
- Team members who came in contact with covid positive people or were secondary contacts, strict quarantine was maintained to contain the spread.
- Most of the brainstorming sessions were conducted on zoom or on google meet to minimise physical proximity as much as possible.
The wetlab aspects were performed in the Teaching and Research complex in three laboratories, including the laboratory of our PI, Dr. Supratim Datta. The general safety observed in experimental design is as follows;
All our experiments belonging to the cloning of the quorum-sensing unit of our project fall under the BSL-1 category. These are performed in the BSL-1 laboratory as per required guidelines. The quorum sensing unit is cloned into E-coli DH5𝛼 and E. coli BL21 and KRX for expression are all BSL-1 bacteria. The chassis organism that we later plan to use is Lactococcus lactis LMG 7930 which also falls under the BSL-1 category of bacteria. A class biosafety cabinet is used for all these experiments.
Pseudomonas aeruginosa was used for our biofilm assay involving DNase I. Since P. aeruginosa falls under the BSL-2 category, all experiments involving the bacteria were done under appropriate guidelines and regulations under a BSL-2 safety cabinet. After the assay, the discarded bacterial cultures were disposed of according to the safety guidelines in a biohazard waste packet to prevent any cross contamination and unintended release of pathogenic bacteria.
In our experiments, we used E. coli DH5𝛼 and E. coli BL21 and KRX as our chassis organism which all fall under the class of BSL-1 microorganisms. In addition, our intended chassis, Lactococcus lactis LMG 7930 which also falls under the BSL-1 category of bacteria. The biofilm degradation assay was performed with P. aeruginosa which falls under the BSL-2 category of microorganisms. All of these microorganisms were handled using the right precautions and discarded safely after usage.
Only safe inserts were expressed. The AgrA,B,C,D circuit produces only one molecule which is the AIP or the autoinducer peptide. The AIP molecule falls within the BSL-1 category and is not documented to cause any harm to humans or any unwarranted harm to the environment. Also DNase I is only documented to degrade biofilms  and do not pose any harm to the environment or to humans if leaked. The rest of the genes only code for receptor molecules and will be destroyed once the GMO lyses. The gene fragments were ordered from twist biosciences and some were supplied by iGEM.
For carrying out our experiments, two different backbone vectors were used - pSB1C3 and PUC19 plasmid backbones. pSB1C3 was obtained from iGEM whereas the PUC19 was obtained from our PI, Dr. Supratim Datta’s lab.
Hazardous chemicals like ethidium bromide were used only under designated areas, handled using appropriate gloves, used only in minor quantities and disposed only in biohazard red bags. In addition all reagents were appropriately handled according to their usage instructions. Any contaminated equipment was disposed of appropriately
Our genetically modified bacteria aims to detect the presence of the target pathogen using the quorum sensing system and then secrete a bacteriocin. This bacteriocin is more specific to the pathogen and serves as an effective alternative for broad spectrum antibiotics. In case our GMO doesn't come in contact with the pathogen, it is equipped with a kill switch so that it lyses itself and does not cause unwarranted contamination via gene transfer.
Endolysin is used to lyse our GMO for the effective release of the bacteriocin Nisin PV which then targets the pathogen Staphylococcus aureus. This not only provides for a method of release of the bacteriocin as said previously but also lyses the GMO. Hence the GMO cannot transfer its genes to any other bacteria residing in the microbiota of the udder and cause unprecedented problems and contamination.
In the unlikely event that our GMO does not come in contact with the pathogen, it is equipped with a kill switch (link to implementation page here) for lysis of the chassis organism. We plan to mutate the thyA gene in our chassis organism using CRISPR Cas9. thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. Mutation of thyA is a recessive lethal mutation that prevents the bacteria from producing any thymidine and has to depend on the surroundings for external supply of thymidine. In the absence of thymidine, the bacteria dies as it cannot synthesize it anymore.
While the bacteria is being grown in the bioreactor, the mutant bacteria is supplemented by thymidine. The vial is then stored in 4 degrees celsius supplemented with thymidine so that the bacterial growth is negligible and the thymidine is unused. When injected into the udder, the bacteria then starts growing utilising the thymidine. In case it comes in contact with the target pathogen, the lysisE7 lyses the chassis GMO and releases the bacteriocin. In case it doesn't come in contact with the pathogen, the dearth of thymidine in the surrounding environment of the udder will kill the chassis. In this manner, thyA mutant lactococcus lactis proves to be an effective chassis.
Our detection kit aims to use the combined cleavage efficiency of Cas13a and Csm6. However this will generate a lot of RNA cleaved products at the end of the procedure. We plan to provide a 10% bleach solution along with our kit so that any RNA residues remaining in the eppendorf tube and not poured into the micropad can be denatured and disposed of properly (link to detection design here). In addition, the micropad is wax-paper based. However it also contains RNA waste. The micropad can be incinerated after use with minimal contribution to gaseous pollution but maximal stop in contamination via cleaved RNA residues.
- Sharma, Komal, and Ankita Pagedar Singh. “Antibiofilm Effect of DNase against Single and Mixed Species Biofilm.” Foods, vol. 7, no. 3, 2018, p. 42., doi:10.3390/foods7030042.
- Ross, P, et al. “Thymidylate Synthase Gene from Lactococcus Lactis as a Genetic Marker: an Alternative to Antibiotic Resistance Genes.” Applied and Environmental Microbiology, vol. 56, no. 7, 1990, pp. 2164–2169., doi:10.1128/aem.56.7.2164-2169.1990.
- “Gootenberg, Jonathan S., et al. “Multiplexed and Portable Nucleic Acid Detection Platform with Cas13, Cas12a, and Csm6.” Science, vol. 360, no. 6387, 2018, pp. 439–444., doi:10.1126/science.aaq0179.