As it should, safety has always been the number one priority to the Vilnius-Lithuania iGEM team. This year we swimmingly extended a great track record set by our predecessors and placed the safety of ourselves and others at the forefront of our projects’ aims. The assistance of accomplished scientists and staff at the Life Sciences Center of Vilnius University (VU LSC) allowed us to fulfill the fundamental task of protecting our environment to the best of our abilities. Careful experiment planning and scrutinous work ethics in the laboratory left no chances for any harm to come to our already delicate nature and the ones relying on its prosperity. The planned experiments were presented in detail to our PI and Dean’s office and they confirmed that our experimental procedure does not have any biosafety concerns in the laboratory.
All laboratories in our country have to be accredited by the Office under the Ministry of Environment in accordance with the requirements of standard LST EN ISO / IEC 17025: 2018. This year our project employed the use of laboratories classified as biosafety level (BSL) 1 and 2. To minimise the risk of cross-contamination, the experiments carried out in BSL-2 laboratory were performed by a single person following strict safety precautions. In addition, the person was supervised by an experienced staff member at all times. All necessary safety and decontamination protocols were obeyed by said person before transitioning between the laboratories.
As per official protocol, all of the laboratory team members underwent detailed safety briefings and training provided by the qualified personnel of VU LSC before any laboratory work had begun. The training included, but was not limited to:
- Proper use of laboratory equipment and devices.
- Briefing on the necessary and proper use of personal protective equipment (PPE).
- Taking the necessary steps towards preventing any fire hazards, including undertaking the official instructions in the event of a fire.
- Following strict hygiene norms (e.g. no food or drinks in the laboratory, long hair must be tied back when doing work of any kind, ensuring proper ventilation of the laboratory spaces, etc.).
- Disposing of biohazardous materials under the controlled supervision of highly trained staff members.
- Steps to be taken and persons to be contacted in the event of spillage of and(or) human exposure to the biohazardous materials.
- Appropriate handling and storage of various hazardous and(or) flammable materials.
In addition, safe work practices were overseen by our PI - Prof. Rolandas Meškys.
Laboratory team members procured or were provided with all the necessary and approved PPE to ensure their safety, including laboratory coats, gloves, eyewear, closed-toe footwear, long-legged trousers, etc. The use of headphones or other hearing-hindering devices were also prohibited. Before entering or leaving the laboratory premises, all necessary sterilisation steps were taken in order to prevent the spread of microorganisms into the environment. Biological safety cabinets were disinfected after each use and deep cleaning was performed at the end of each week.
To prevent cross-contamination of laboratory surfaces and biological safety cabinets proper use of various disinfecting sprays and aseptic technique was employed.
The nature of our project demanded involvement of a single species belonging to the risk group 2 - Klebsiella pneumonia (strain KV-3), however we managed to reduce any possible risk of human exposure to a minimum. In fact, the bacteria were only used at the absolutely-necessary stages of project development for proof-of-concept purposes and the final product would not contain or promote the proliferation of any harmful bacteria nor be adverse to the environment. Our PI provided the Klebsiella pneumonia along with all the necessary instructions for a safe work approach. As mentioned before, only a single person was working with K. pneumoniae in a BSL-2 rated laboratory while also being supervised. The person had no known immunodeficiency and no chronic diseases, which could possibly make him susceptible to the infection. On the days of the experiments the person did not come in contact with any of the other team members.
All other microorganisms used are classified as belonging to risk group 1 and were handled in a BSL-1 rated laboratory. These included well-characterized and non-pathogenic Escherichia coli strains (E. coli BL21 (DE3), Rosetta (DE3), Arctic Express (DE3), DH5α, DH10B, and ER2566), already present in our lab inventory and used as a chassis for most of our parts.
The remaining part was expressed in a Saccharomyces cerevisiae (strains 214, 214Δpep, Gcn2, 214mn 1D and Fh4c). The strains were kindly donated to us by VU BTI EGIS laboratory and transported in an appropriate hermetically sealed packaging, thus no risk of release to the environment was posed. Additionally, when working with the yeast strain a separate biological safety cabinet was used to eliminate any possibility of cross contamination with the other microorganism species.
The last three risk group 1 organisms we used belonged to the family of Flavobacteriaceae: Flavobacterium branchiophilum (strain 24789), Flavobacterium psychrophilum (strain 18644), Flavobacterium columnare (strain 3660). All of these microorganisms were obtained from The Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH. These organisms are known to infect animals, however every precaution was taken so as to not release these organisms into the wild.
In its entirety, the project was divided into three main subprojects: detection, treatment and prevention.
This part of the project was nominated with the task of developing a pathogenic bacteria detection kit based on isothermal helicase-dependent amplification (HDA) and lateral flow assay (LFA) methods. The kit contains no cells and poses no threat of bacterial contamination. The HDA enzymes are also harmless. As long as it would be handled using the instructions provided and disposed of appropriately it presents no threat to the environment1.
Currently, this is merely a laboratory-contained exercise intended for a scientific advancement of the future treatment capabilities and other applications of GMOs. Regardless, no proteins or other materials that could be released by our designed transfer bacteria are known to cause any harm to the environment nor were they exposed to it. At no point was it considered to apply the use of this treatment method in natura. Every precaution was made to prevent the release of GMOs into the environment as per the rules of iGEM and regulations of VU LSC. In an extremely unlikely case of environmental release, the GMOs were equipped with two separate kill-switch mechanisms in order to prevent their spread.
The idea of preventing fish disease was to develop an environmentally friendly edible vaccine. Thus, by design, none of the materials it contains are harmful to the aquatic environment or the environment in general. The vaccine contains no cells and only their protein products, carefully purified using protein chromatography and dialysis techniques, which are known to prevent bacterial contamination2.
No parts used in the project’s development are known to be toxic to humans, animals or plants, thus no risk was placed on the persons working with the cellular products and only the target bacteria would be affected.
Some experimental techniques required the use of UV light or usage of compounds, requiring additional safety protocols. All necessary precautions were taken to prevent harm.
UV light: UV light was used to sterilize biological safety cabinets and the equipment contained in them, as well as for DNA imaging in agarose gels needs. Our team members made certain to wear necessary PPE. Due to very few personal close-up encounters with UV light and having taken safety precautions, we completely minimized the possibility of experiencing UV damage and the health risks concerning it. Also, to minimize the contact time with the UV light, we introduced blue light transilluminators to common laboratory procedures.
Ethidium Bromide: This compound is a potent mutagen and is moderately toxic after acute exposure. Therefore, the lab team took it upon themselves to familiarize themselves with the appropriate procedures that are required to handle cases of spills or acute exposures. These procedures included working in a functioning fume hood and wearing personal protective equipment (PPE) to minimize the chance of exposure, as well as extensively studying the material safety data sheet (MSDS) for Ethidium Bromide. We disposed of the product in accord with the disposal protocols and the state safety regulations.
Antibiotics: We used a few main antibiotics for the selection procedure. According to the material safety data sheet (MSDS) for ampicillin, chloramphenicol, and kanamycin, these compounds are known to have hazardous effects at high concentrations and both acute and chronic exposure to humans via inhalation, ingestion, or contact with eyes. Therefore, the team wore personal protective equipment (PPE) when handling the compound, studied the safety protocols, and were equipped with the necessary information to handle spills, acute exposures, and disposed of the product in accord with the disposal protocols as well as the state safety regulation
Acrylamide: In a laboratory setting, acrylamide is a widely used compound, necessary for many research procedures. Unfortunately in its pure form it is highly toxic and mutagenic. As with any other reagent, however, our team took all necessary steps in ensuring its safe use by strictly adhering to the manufacturer’s safety data sheet along with appropriate disposal of the gel.
Once again it should be reiterated that all of the organisms, their products and other reagents were handled conscientiously and by abiding all the pertinent safety guidelines provided by their relevant providers. At no point did our team members deviate from the strict safety standards and policies presented to us by the VU LSC, iGEM and the suppliers.
- Wingfield, P. T. Overview of the purification of recombinant proteins. Current protocols in protein science 80, 6.1.1-6.1.35 (2015).
- Nanotechnologies - Part 2: Guide to safe handling and disposal of manufactured nanomaterials (2013).