Introduction

Since Lithuanian teams began participating in the iGEM competition, safety has always been the top priority. This year was no exception, and our Vilnius-Lithuania 2024 iGEM team strictly adhered to safety protocols throughout the project, ensuring all experiments were conducted with rigorous attention to biosafety standards. Our experiments and methods were reviewed and approved by our Principal Investigator (PI) and qualified scientists at the Vilnius University Life Sciences Center (VU LSC), confirming that our project does not pose any safety concerns in the laboratory. To maintain a safe and secure research environment, all our work was supervised by experienced personnel, including our PI. Adhering to the measures listed below ensured the highest biosafety levels throughout our project.

We regularly consult the iGEM Safety and Security Committee to ensure compliance with safety requirements, and no prohibited activities are part of our project.

Safe project design and used microorganisms

Our project involves engineering strains of Escherichia coli to produce environmentally friendly polysaccharide glue under controllable conditions. We are transferring pathways from organisms like Caulobacter crescentus and Hirschia baltica to achieve this. These organisms and parts are all non-hazardous and conform to iGEM guidelines.

The project design does not involve activities (like working with animals or animal samples, using human samples such as blood or other bodily specimens, or using environmental samples and gene drives) that require advance permission from iGEM. In addition, when designing our project, we took care to avoid increasing risks related to antimicrobial resistance. Also, our work is fully covered by the iGEM White List, and no organisms or parts outside of this list are used. 

The experiments involved gene cloning and protein expression using non-pathogenic, well-characterized strains of Escherichia coli: Mach1, DH5-alpha, dh10b, KRX (DE3), BL21 (DE3), C41 (DE3), Rosseta (DE3)pLysS, HMS174(DE3), B3784 - WecA - E. coli BW25113 ΔwecA, B3784 - WecB - E. coli BW25113 ΔwecB, B3785 - WecF - E. coli BW25113 ΔwecF, B4481 - WecG - E. coli BW25113 ΔwecG, B3794 - WzzE - E. coli BW25113 ΔwzzE.

As part of the project, we also worked with Caulobacter crescentus (Caulobacter vibroides) CB2 (DSM 4727, ATCC 15252), Caulobacter crescentus (Caulobacter vibroides) CB2A (DSM 25117), and Hirschia baltica (DSM 5838, ATCC 49814). These organisms naturally occur in the environment, are classified as BSL-1, and pose no risk to human health or their surroundings.

Our engineered organisms are designed for controlled use, with no plans for release beyond laboratory containment. Extensive safety testing would be conducted if the project were to progress toward real-world applications, such as using glue for medical patches, and no live organisms would be part of the final product.

Safe lab work and experiments

The project was carried out in a Biosafety Level 1 (BSL-1) laboratory, which complies with the hygiene, fire, and work safety standards required by the Office under the Ministry of Environment, following the LST EN ISO / IEC 17025: 2018 standard. All team members wore protective lab coats, gloves, and eye protection. Experiments with bacteria were conducted within a biosafety cabinet to prevent contamination.

Before starting any lab work, all team members underwent comprehensive safety training covering chemical and biosafety, proper use of biosafety cabinets and equipment, emergency response, and accident reporting. This training included teaching the proper use of laboratory equipment, hygiene protocols, handling hazardous substances, and disposing of these substances following regulations.

Our team works under the supervision of our PI, Rolandas Meškys, and consults regularly with biosafety officers and other experts. Risk management includes: routine disinfection and sterilization of workspaces, monitoring and documenting any accidents, and adherening to lab access rules, biosafety level protocols, and emergency procedures.

Safe use of harmful reagents and procedures

Our project involves hazardous chemicals like carcinogens (Congo red), mutagens (ethidium bromide, phenol) acrylamide, highly flammable chemicals (ethanol, isopropanol, methanol), acids and corrosive chemicals (HCl, NaOH), antibiotics (Kanamycin, Carbenicillin, Ampicillin, Chloramphenicol, Streptomycin) and other hazardous chemicals (Sodium dodecyl sulfate (SDS), Acrylamide, TEMED, APS).

Proper PPE (lab coats, gloves, eye protection) and fume hoods are used, and all chemicals are handled and disposed of according to safety guidelines and safety data sheet protocols. UV light is used for sterilization, and antibiotics are used for selecting transformants, with strict safety measures in place.

Environmental safety

To ensure that no hazardous materials or organisms leave the laboratory:

• All lab waste was treated following faculty protocols.
• Tools and materials were autoclaved after each use.
• Workspaces and clothing were disinfected regularly.
• The engineered organisms we worked with could not survive outside controlled environments, minimizing the environmental risk.

Data security

Although our project involves some data collection related to human practices, we do not collect data from human subjects that require formal ethical approval. Any information gathered is handled in compliance with national and institutional guidelines.

During human practices activities, we collected data in compliance with the General Data Protection Regulation (GDPR). Our mobile phone game, Bacteriomania, collected only anonymized data using cookies, as outlined in our privacy policy. Personal data collected through surveys and other activities was handled with strict confidentiality, and appropriate security measures were taken to protect the information.