Overview

JetroEco, as an initiative to make commercial aviation more sustainable and eco-friendly, would be of little purpose if the safety of the solution and the stakeholders were not adequately ensured. Thus, it was essential to look into all aspects of the processes and workspace practices to make sure safety is prioritised.

Project Design

The Microorganism

JetroEco bases itself on the organism Yarrowia lipolytica, a spore-forming oleaginous yeast, not only for metabolic engineering potential but also for the low risk it poses for human safety and the environment.

Yarrowia lipolytica is classified as a BSL-1 organism by the United States Public Health Service and is granted the Generally Recognised As Safe (GRAS) status by the U.S. Food and Drug Administration (USDA). It has also been concluded that Y. lipolytica has the equivalent level of non-pathogenicity as S. cerevisiae^[1]. The organism’s BSL-1 status and its inability to produce toxins make it suitable for use in large-scale production facilities under conditions not exceeding the Good Industrial Large-Scale Practice (GILSP) level of physical containment.

The Approval

Before we started our experiments, it was vital to get the approval of our institute’s biosafety committee as per the revised Regulations and Guidelines for Recombinant DNA Research and Biocontainment, 2017. So, we had meetings with our institute’s biosafety committee head Dr. Sunish Radhakrishnan and filled out the respective Biosafety Clearance forms with the help of our primary PI Prof. Saikrishnan Kayarat. Only after this approval did we start our experiments.

General Lab Safety

Owing to Y. lipolytica being a Risk Group-1 organism^[2], the JetroEco project would primarily allow working in BSL-1 workspaces. As a part of this, the wet lab team had to adhere to certain guidelines and precautions while working on biological organisms:

• Smoking, eating and drinking were not allowed in the laboratory.
• It is mandatory to wear appropriate protective gear. Long hair should be tied back.
• Mouth pipetting is prohibited.
• Washing of hands regularly is necessary.
• Wearing gloves is mandatory, except for when operating an electric/gas burner.
• Protection glasses should be worn, more so when using UV light.

It is also important to stay vigilant while working with the various instruments in labs, to prevent any cases of harm while operating:

• Centrifuge: Potential risk of injury. Always ensure that the centrifuge is balanced and the lid is in place before running.
• Electrophoresis Tank: Potential risk of electrocution. Always keep the safety lid on the cell before switching it on.
• Water Baths: Potential risk of electrocution or burns. Always check if the bath apparatus is in good condition before use.
• Ethidium Bromide: Potential contamination and exposure to a CMR substance. All such use should be confined to a specific workspace, with an appropriate disposal system.
• Chemical Fumehood: Possible case of inhaling toxic chemicals.

The team also ensured that the waste generated during the wet lab cycle was disposed off in an appropriate manner:

• Common Waste.
• Biological Waste: Sterilized with bleach and ethanol before disposal
• EtBr Waste: Discarded in separate containers and disposed of independently.

In order to work in a sterile environment, we conducted our experiments in biosafety cabinets (BSCs). These cabinets were disinfected before and after use with ethanol and were sterilized with UV light after the completion of the protocol within them.

We have also always conducted all of our culture-related experiments at our institute in a BSL-2 hood, as advised by our mentors, and we have meticulously ensured that all of our non-hood experiments were conducted with consideration for all safety issues. At all times during the experiments we conducted, we wore gloves, masks, protective gloves, and coats. We have separate areas set aside for EtBr to run our agarose gels. Additionally, we have distinct bins for the dumping of plastic, liquid, and biological sample garbage. We also had separate trash cans for both liquid and solid EtBr-contaminated products. At least one of our mentors oversaw the execution of each of our experiments.

Biosafety Meeting

In collaboration with ICT-Mumbai, we organised a biosafety webinar on 26 July which was hosted by Dr. Shamlan S. Reshamwala, Assistant Professor, Center for Energy Biosciences, ICT Mumbai.

In this webinar, an introduction and overview of why our designed GMOs should be contained within the reaction containers was given. Alongside this, we also learned about many strategies for preventing the accidental release of our GMOs. Such strategies included the use of auxotrophies (synthetic or natural), kill switches, ligand-dependent stability, and induced environmental sensitivity. We learned about the merits and demerits of each strategy and the appropriate situations to use of them.

HGT and Respective Prevention

As with any other biologically engineered organism, the risk of horizontal gene transfer (HGT) is prevalent in our modified Yarrowia lipolytica. However, phylogenetic studies analysing such gene transfers have shown that horizontal gene transfers (especially the transkingdom ones) are very rare in yeasts, with no such transfer event has occurred with respect too Y. lipolytica in the last two decades. Thus, regular disinfection protocols are sufficient enough should an escape event of the organism occur from its industrial container.