Ensuring safety is of utmost importance for scientists conducting genetic and microbiological studies. At Estonia-TUIT, our objective is to investigate the potential of using S. cerevisiae to produce siRNAs that would support bees in fighting against Deformed Wing Virus. From the initial project design concepts to the completion of the final experiments, we adhere to three fundamental components of biosafety: laboratory practices and techniques, safety equipment, and facility design (Looby et al., 2022).

Laboratory practice and technique

Prior to commencing any laboratory work, our team underwent an intensive and comprehensive introductory safety course conducted by our Principal Investigators (PIs), instructors, and advisors. This program included both theoretical and practical exercises tailored to the context of our research plans, along with the mandatory biosafety training. All the laboratory work was conducted according to the institute safety guidelines (Workplace Safety Instructions | Tartu Ülikool). Our team works only with non-pathogenic organisms belonging to the White List.

Once we started our practical work in the laboratory, our instructors consistently provided guidance and assistance throughout our experimental work. They were readily available to address any queries we had regarding the procedures and ensured that we adhered to safety protocols to maintain a safe working environment.

Safety equipment

Safety is not solely dependent on techniques and practices; it also hinges on the equipment we use during experiments. At the Institute of Technology of the University of Tartu, we have the advantage of access to state-of-the-art, certified equipment. Our work was carried out in a Biosafety Level 1 laboratory, where we adhere to strict safety protocols. This includes wearing certified protective gloves, lab coats, and protective eyewear. To prevent any potential hazards, we ensure that long hair is securely tied up, and any loose or overhanging clothing is stored in designated lockers or wardrobes. When handling UV light sources or liquid nitrogen, we used the appropriate eye protection and gloves to ensure safety at all times.

Facility design

The wet lab activities were carried out at the Institute of Technology of the University of Tartu. As part of our introductory safety course, we received comprehensive training on safety protocols covering fire, chemicals, machinery, and medical regulations. We were shown and had to remember the exact locations of fire extinguishers, fire blankets, chemical showers, and first aid stations and comply with Institute of Technology Fire Safety Rules (Workplace Safety Instructions | Tartu Ülikool).

Project design

Our team has set a goal to preserve and limit the effect of the DWV on the populations of honey bees. We looked into the possibility of using the innate viral protection of the bee to alleviate the introduction of any external enzymes. Honey bees produce Dicer and Argonaute proteins to mediate RNA interference as an antiviral response. As we chose RNA interference system to target the virus, we could target specifically the viral RNA. We carried out computational analysis to look for possible off-target effects of the studied siRNAs in mites, bees and human transcriptome. The detailed results, including potential off-targets and their consequences, are presented in Table 1. The computational analysis did not find any strong unintended targets. We have also consulted with Eva Žuzinaite, Associate Prof. from the University of Tartu and iGEM safety committee to ensure our experiments with siRNA are safe to conduct. In addition to finding off-targets and their consequences we also made an illustration depicting which locus on which chromosome is affected by it, Fig 1.

Figure 1. Humon karyography with affected locusts showed in color. Beige color stands for shRNA V1, light blue - shRNA v2, pink shRNA V3, green - shRNA v4, yellow - shRNA v5, red - shRNA v6, blue - shRNA V7 and V8, orange - shRNA V9, purple - shRNA V10

Further, with an emphasis on biosecurity, we set out to design a method to allow testing of anti-viral siRNAs in a safe model organism, Saccharomyces cerevisiae. Using this testing system allows us to go further in the project without having to work with viable DWV.

In the current design, we use pRS yeast-bacteria shuttle vectors that contain an ampicillin resistance gene for E. coli. Using yeast with these plasmids as a food supplement could release this antibiotic resistance gene to the environment. However, at the current stage, we carry out all experiments in a contained lab. When moving the project to the next steps, a marker-free approach, for example CRISPR, should be used for shRNA expression.