Introduction

The Vilnius-Lithuania iGEM team believes that safety is an integral part of science. It strictly defines what is safe, moral and ethical, protects sensitive groups and the environment. For this reason, the safety aspect was analyzed from both perspectives – inside and outside the laboratory. For a detailed overview, please check our safety form.

Lab safety

All laboratories in our country have to be accredited by the Office under the Ministry of Environment following the standard LST EN ISO / IEC 17025: 2018 [2]. This year the project was conducted in the BSL-1 laboratory, where we fulfilled all requirements for hygiene, fire, and work safety. Furthermore, team members wore lab coats, gloves, and eye-protection glasses during the experiments. Work with bacteria was only performed in the biosafety cabinet.

Before any laboratory work started, the team received safety training which mainly included: appropriate use of equipment, following hygiene rules in the laboratory, work with hazardous substances, and their proper disposal. Also, experiments were presented in detail for our PI prof. Rolandas Meškys and Dean’s office. They confirmed that our experimental procedures do not have any biosafety concerns.

Microorganisms review

Protein expression for the diagnostic test was performed in non-pathogenic and well-characterized Escherichia coli strains: BL21 (DE3), Rosetta (DE3, gami), HMS174, Aurora, XL1-Blue, DH5 alpha, TG1, JM109. They were already present in our inventory. As chasses, Escherichia coli Nissle 1917 (kindly provided by Pharma-Zentrale GmbH) and Lactobacillus casei BL23 (kindly supplied by the Vilnius University Life Sciences Center Department of Biological DNA Modification) were used. These strains are probiotic and are discovered in the environment naturally [3, 4]. They do not possess serious illnesses or other problems to the people or the environment and belong to the BSL-1 organisms [5, 6].

Experiments

The prevention tool (probiotics) was created by transferring the naringenin biosynthesis pathway to the probiotic bacteria, which naturally occur in the human intestine and pose no danger. A VapXD kill-switch was designed to prevent GMO probiotics from escaping the human body. While working with GMOs, team members took every precaution as disinfection and sterilization of clothing to avoid contaminating the environment with GMOs. Also, all biocontaminants were submitted for autoclaving and disposal.

A diagnostic test was developed with recombinant Entamoeba histolytica proteins (cysteine proteinase 5 and pyruvate, phosphate dikinase), which are not pathogenic by themselves. Therefore, as a diagnostics proof of concept, we planned to use blood. Concerning this matter, we contacted the Lithuanian Bioethics Committee. They had the meeting and decided that no permission was needed.

Harmful reagents and procedures

Some planned experiments had to be done by using harmful reagents or procedures. Team members followed all rules and guidelines to prevent harm for themselves or the environment as it was taught in the safety training.

UV light was used to sterilize the biosafety cabinets and equipment inside them. All safety measures were taken to avoid direct exposure to UV light. For agarose gels’ visualization gel imaging system was used, and standard laboratory procedures were performed with blue light transilluminator.

Antibiotics were used for transformants selection. At high doses, they can be toxic when inhaled, ingested, or in contact with the eyes. Antibiotics were handled with appropriate safety measures and disposed of according to the disposal protocols.

Ethidium Bromide was needed for the agarose gel visualization. As this chemical is a potent mutagen and is moderately toxic after acute exposure, team members followed all requirements to stay safe according to the material safety data sheet (MSDS).

PDA synthesis contains some of the cancerogenic materials 10,12-Tricosadiynoic acid (TCDA), 10,12-pentacosadiynoic acid (PCDA), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC-HCl), Ethanolamine, N-hydroxysuccinimide (NHS), Dimethyl-2-(dimethylphosphino)ethylphosphine (DMPE). Whole-body and head protection was used to ensure safe usage. Synthesis was done in a fume hood.

Other hazardous chemicals such as Triton X-100, nitro-cellulose, Pb salts, HCl, MeOH, Cu salts, NaOH, KOH, TRIS-HCl, TRIS-Base, Phosphate buffer, etc. were used while taking every precaution needed and disposing of the waste as it is required by the MSDS.

Data security

During the execution of three human practices activities, we have received data associated with people. We followed The General Data Protection Regulation [1] and the measures that we have taken in a particular context are explained further on.

Augmented reality project “6th SynBio Sense” is a website that uses cookies. As this is an ongoing project that the Vilnius-Lithuania iGEM 2020 team started, the privacy policy was already set. In general, the policy includes information about what data is being collected, how it is used, and contacts to address any questions. The website does not collect names, locations, e-mails of the users, or any other information that could be used to identify a person. Data is only collected via cookies. They are two types:

• Essential - any user is obliged to agree to this cookie being used. Through this cookie, the website collects information about the selection of language and type of font (there is an alternative font for dyslexia).
• Statistical - optional “Google Analytics” cookies that provide information about website users. No information that could identify a person is not collected.

Complete “6th SynBio Sense” privacy policy can be accessed here.

The Learning about Life Sciences survey collected no personal information to link a person to their answers. Collected data contained age, sex, residence (city, town, small-town), area of specialization; this information was used to characterize the sample.

During four Sense Lab cycle activities (touch, taste, smell, hearing), we collected this personal data: name and surname, telephone number, e-mail, age. As this is sensitive information, the information was tackled with the highest regard for security. Google forms were used as a tool, and each of their descriptions information about data collection and usage was presented.

COVID-19 associated safety measures

Team members followed all the government and university guidelines based on the pandemic situation in the country. For the first five months, teams' meetings were present online, and activities in person were allowed only if inevitable. COVID-19 tests were performed regularly until the whole team got vaccinated.

When working in the lab began, a 2-meter distance was kept between members, and attendance was recorded. What is more, the usage of lab equipment was registered. All working surfaces were cleaned with disinfectant fluid before and after work.

Our team spent a lot of time responsibly planning public awareness events to consider all necessary safety measures. We followed the highest safety standards and required participants to participate only with a National certificate, which proves that a person who has a negative COVID-19 test has been vaccinated against COVID-19 or has recovered from COVID-19. In each of the events, we provided disinfectant, ventilated premises.