As in previous years, the principal value of the Vilnius-Lithuania iGEM team is safety. Every hazard and condition leading to physical, psychological, or material harm is controlled to preserve the well-being of the team and the environment. Throughout the project, our team responsibly planned the experiments considering all possible safety issues beforehand. The help of well-experienced laboratory staff members helped us to analyze all the potential security risks and their counter measurements. The planned experiments were presented in detail to our PI, and it was confirmed that our experimental procedure does not have any significant biosafety concerns in the laboratory.

All laboratories in our country must be accredited by the Office under the Ministry of Environment following the standard LST EN ISO / IEC 17025: 2018. This year, our project was done in a laboratory classified as biosafety level 1 (BSL-1). All requirements for hygiene, fire hazard and work safety were fulfilled. Team members responsibly followed the safety rules and wore personal protective equipment (PPE), such as lab coats, gloves, goggles, and - in some cases - respirators. We ensured that work including bacteria would be performed only in a biosafety cabinet, which was disinfected before and after each use, and sterilized under UV light.

Before starting work in the laboratory, our team received safety training, which focused mainly on the correct use of equipment, hygiene rules, and safe usage and disposal of hazardous chemicals.

Protein expression was performed in non-pathogenic and well-characterized Escherichia coli strains: BL21 (DE3), HSM174, KRX, ArcticExpress (DE3), and DH5α. All these strains are safe to work with in the BSL-1 laboratory and were kindly provided to us by laboratories of the Vilnius University Life Sciences Center.

The nanoplastic detection system is based on plastic binding peptides, which are considered to be antimicrobial. Our team members took every precaution to avoid contaminating the environment with GMOs. Therefore we sterilized and disinfected our workspace and clothing regularly. All biocontaminants were sterilized and disposed of safely.

Our detection system is designed to use purified proteins without bacteria, and the test should be used in a closed system in a laboratory setting. This way, we ensure that our created synthetic peptides will not leak out of a laboratory setting, where safety is highly regulated.

Some experiments could not have been done without the use of UV light or the usage of harmful reagents. Our team followed all requirements to ensure safety for themselves, others, and the environment.

UV light: it was used to sterilize biological safety cabinets and the equipment inside them, as well as for the agarose gel visualization. Standard laboratory procedures were performed using blue light transilluminator instead of UV light where possible. Team members followed safety rules, such as wearing protective eyewear and gloves to minimize the possibility of experiencing UV damage and the health risks concerning it.

Antibiotics: they were used for the selection procedure after transformation. According to the safety data sheets (SDS), kanamycin, carbenicillin, ampicillin, and chloramphenicol are acutely toxic and have hazardous effects when exposed to skin and eyes or inhaled at high concentrations. Therefore, our team members made certain to study SDS to learn how to store and handle these materials.

Ethidium Bromide: it was used for agarose gel staining. Ethidium bromide is known to be moderately toxic after acute exposure and potentially mutagenic. Therefore, our team carefully studied the SDS for this chemical. All team members wore the required PPE and made sure to dispose of this chemical according to the disposal protocols and state safety regulations.

Acrylamide: it was used for the SDS-PAGE gels. Acrylamide is acutely toxic and mutagenic. As for other materials, our lab members studied the safety rules for this material, learned how to dispose of this chemical. When working with acrylamide powder, PPE and a fume hood were used.

Other: all hazardous chemicals, which were not mentioned above, such as Triton X-100, nitrocellulose, HCl, copper salts, NaOH, Tris-HCl, Tris-Base, etc. were used and disposed of according to the recommendations provided in the SDS.

To summarise, the team members strictly followed the safety standards, policies, and guidelines provided to us by the Vilnius University Life Sciences Center, iGEM HQ, and product suppliers.

Human exposure to nanoplastics through inhalation and ingestion has raised concerns about their potential adverse health effects. The toxicity of inhaled and exposed nanoplastic particles is not well characterised in part due to the complexity of their chemical compositions, size, and shape. Therefore, it is recommended to handle them as potentially hazardous material.

Since nanoplastics may cause potentially hazardous effects, we followed strict rules to make sure we were keeping ourselves safe during all experimental procedures:

• 1) We were thoroughly instructed by our Life Sciences center staff and an expert working with nanoplastic how to handle nanoparticles and how to protect ourselves. This included thorough SDS analysis, handling instructions.
• 2) We were informed about the 4 possible routes of workplace exposure to nanoparticles:
• Skin absorption: nanoparticles have been shown to migrate through skin and circulate in the body.
• Injection: accidental injection is a potential route of exposure.
• Ingestion: when ingested, nanoparticles may be absorbed and transported within the circulatory system.
• Inhalation: because of their small size, nanoparticles can penetrate deep into the lungs and other organs.
• 3) We wore appropriate PPE:
• Double nitrile gloves. We used double gloves in order to place outer gloves in a sealed bag to prevent nanoplastic particles to become airborne after work.
• Lab coat, which is fully extended to the wrist.
• Safety goggles.
• A respirator or a face shield to prevent potential inhalation.
• 4) Nanoparticles were handled in a form that is not easily made airborne, such as in a solution or on a substrate.
• 5) Experiments with nanoparticles were performed in a specially designed "NanoBox", which served as a workplace, when handling nanoplastic materials.
• 6) Eating, drinking, chewing gum or handling contact lenses was not allowed.
• 7) All nanoplastic waste was treated as hazardous and disposed following the same rules as hazardous chemicals.

We believe that careful handling of nanoplastic helped us to keep safe and may set a standard for future iGEM teams, who will be trying to work with specific nanoplastic materials for their projects.

At no point did our team members deviate from the strict safety standards and policies presented to us.

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