Safety and security play a very important role in performing responsible research. Safety does not always mean laboratory-related safety; it also considers the safety and security regulations related to Human Practices.
Lab Safety
The first thing our team did before starting the wet lab experiments was to go through the safety protocols of iGEM and the ones mentioned by the Department of Biotechnology, Government of India. Each and every member of the team underwent safety training related to safe lab practices and biosafety protocols facilitated by the IISER Pune Biosafety Committee. Our PIs (Dr. Saikrishnan Kayarat and Dr. Mridula Nambiar) and our project mentors helped us do a risk assessment of our experiments during the experimental planning phase.
A few of our teammates also received lab safety training related to handling the Azospirillum species from our mentors in BHU and Dr. Anil Kumar Tripathi, who has decades of experience in working with Azospirillum.
We adhered to the iGEM safety and security policies throughout the cycle. While working in the lab, we used appropriate personal protective equipment such as lab coats, nitrile gloves, masks, shoes and eye goggles.
All the labs we have worked in follow BSL1 biosafety protocols.
Waste Disposal
The waste produced in the laboratory was segregated into biological solid/plastic waste, biological liquid material waste, and other solid waste.
Biological solid/plastic waste is collected in a bag and autoclaved for decontamination before being incinerated. Biological liquid waste is discarded in a container and treated with 10% bleach for disinfection, and kept overnight before being discarded. The other solid wastes are discarded with other waste.
Ethidium bromide, which is used to run agarose gels and SDS page gels, is carcinogenic and thus, gloves, gels, and other objects with EtBr have to be disposed of properly. Gels and gloves containing EtBr are kept in a ziplock bag, discarded separately from other wastes and then incinerated.
We also took care to discard organic solvents and their plastic containers separately from other liquid solvents.
Project Safety
Movement of genetic material between two organisms can happen via vertical transfer (which is the transmission of genetic material from parent to offspring) and horizontal transfer. Transformation, transduction, and conjugation are the three well-understood mechanisms of horizontal gene transfer (HGT) which plays a major role in fueling pathogen evolution by the transfer of antibiotic resistant genes. While introducing GMOs in the environment, HGT is an important thing to consider from the biosafety point of view because spread of the antibiotic resistance genes to human pathogens can cause a surge in diseases that are difficult to tackle.
Our chassis, Azospirillum brasilense Sp7, is a plant growth promoting rhizobacteria commonly used in fertilisers. Our project proposes that our genetically modified Azospirillum brasilense Sp7 is a better biofertiliser as it can convert the excess ACC produced during waterlogging stress into by-products. Administering our chassis as a biofertiliser in the soil without any biocontainment strategy can be risky as it can potentially lead to the transfer of unwanted genes in the soil bacteria.
We have always been thinking of biosafety since the start of our project. The first step of our wet lab was to introduce our gene of interest (acdS) under a hypoxic promoter in the genome of our chassis instead of a plasmid. This is because the rate of gene transfer by HGT is lower if the gene is present in the genome over a plasmid. However, we did not get enough time to implement the genomic integration part of our project.
We also planned to overexpress exopolysaccharides (EPS) in our chassis to enhance its plant growth promoting ability as EPS is a bio sponge that promotes nutrient and water absorption in plants. The EPS genes will be artificially introduced into our chassis with the help of a plasmid which will contain antibiotic resistance genes for screening. We cannot afford to allow horizontal gene transfer of these antibiotic resistance genes in soil bacteria. Hence, it was absolutely essential that we have a biocontainment strategy for our EPS plan. To know how our kill switch works please read the Implementation Page!
As a part of our future experiments, we are planning to integrate the genetic circuit with acdS gene into the genome of our chassis, Azospirillum brasilense. This process is known as genome integration and reduces the chances of HGT.
All the organisms and parts used in our experiment or that will be used in future are present in the White List and are risk group 1 organisms .
Also, we plan to approach the Genetic Engineering Appraisal Committee (GEAC) get regulatory approval for field trials of our genetically modified biofertiliser and engage the help of legal and scientific experts to check if our product and activities follow the Fertiliser Control Order, 1985 and the Environment Protection Act,1986.
Safety in Experiments
We are working with both Escherichia coli and Azospirillum sp. for our project.
The strains used/will be used for E.coli are:
- DH5-⍺
- BL21-DE3
- NEB Turbo
- s17.1
The species and strains used/will be used for Azospirillum are:
- Azospirillum brasilense sp7 (our chassis)
- Azospirillum brasilense Cd
- Azospirillum brasilense Sp245
- Azospirillum oryzae TNCSF Aa13
- Azospirillum lipoferum 4B
A. brasilense and A. lipoferum have been used to generate growth curves.
We have cloned the acdS gene in E. coli DH5-⍺ plasmid for proof of concept.
As mentioned before, we plan to introduce the acdS gene (our source of the acdS gene is A. lipoferum) into A. brasilense sp7 (via genome integration) and E. coli BL21-DE3 (plasmid transformation). Both organisms will be engineered to have an alcohol-inducible exaA promoter.
We will be testing the efficacy of the alcohol-induced promoter, exaA, under hypoxic conditions in E.coli BL21-DE3. We will also be characterising the E.coli hypoxia promoter fdhF in Azospirillum brasilense sp7.
We have ensured that all our experiments and activities are on the iGEM white list and have the approval of the IISER Pune Biosafety Committee.
Human Practices - Safety and Security
While engaging with our stakeholders to get inputs on our project—surveys, talks, meetings, education—we ensured that all the activities were socially responsible and followed the necessary guidelines set by the IISER Pune Ethics committee and iGEM.
Dr. Aurnab Ghose (who heads the IISER Pune Ethics Committee) was involved in making sure all our surveys followed the Institutional guidelines. We carried out the surveys and made the results public after getting approval from the ethics committee.
Collecting informed consent
We made sure each and every person filling in the surveys knew that:
- Information we are collecting and its purpose.
- Participation is voluntary, and they can stop contributing at any point without any consequences.
- The data collected will be anonymised.
All photos and recordings of our meetings with stakeholders and experts were taken and put up on our wiki after taking permission from the participants.
All the information about surveys put up on our wiki has been sanctioned by the institutional ethics committee.
Apart from all these, we also attended the Values and Risks workshop organised by iGEM which helped us a lot in learning how to adhere to the safety policies of iGEM and strengthen our surveys, interviews, webinars and human practices.
References
[1]https://www.atcc.org/products/29729
[2]https://biosafety.icar.gov.in/category/indianbiosafetyguidance/
[3]https://biosafety.icar.gov.in/wp-content/uploads/2015/11/The-Environment-Act-1986.pdf
[4]http://agriportal.cg.nic.in/beejnigam/ActandRules/Fertiliser(Control)Order,1985.pdf
