Safety is a crucial component of synthetic biology and the iGEM competition. Whether it relates to the methods and materials used for the experiments, the lab conditions, the parts and microorganisms used, or even the biological design itself, we believe that assuring safety should be a priority for every scientist. On this page, we have attempted to compile all the aspects of our project where we strived to maintain this principle throughout our project.

Ensuring laboratory safety is essential for avoiding any potential accidents. Since our wet lab department comprises of undergraduate students, close supervision was mandatory for conducting experiments in the lab. Our instructor and advisors provided that supervision. Also, before our members started the experiments, a lab training was held for our members to familiarize themselves with the space. In this training, our teams learned about the safety rules and protocols within the lab while also helping to handle laboratory equipment, such as autoclaves and laminar flow hoods. Once more, our instructor, advisors, and fellow researchers within our department extended their guidance and support during this training phase.

DH5-alpha

For some of our experiments, we decided to utilize the common-lab bacterium Escherichia coli. More specifically, we used the strain DH5-alpha, a derivative from the K-12 strain for the creation of our design’s genetic constructs. Although extensive research on this bacterium has been conducted and the risk for an adverse outcome is minimal, we remained committed to implementing precautionary measures within the lab, preventing potential contact by wearing gloves and coats, routinely sanitizing our equipment and workspace, and maintaining hygiene.

This strain was kindly provided to us by Plant Env. Lab of the Department of Biochemistry and Biotechnology, University of Thessaly

BL21 (DE3)

Strain E.coli BL21-DE3 was one of the chassis organisms we incorporated in our synthetic consortium. The specific strain, derivative of the E. coli B strain, was tasked with expressing our designed constructs for producing the essential proteins for detoxifying Olive Mill Wastewater (OMW), generating and secreting free fatty acids (FFAs). Given its pivotal role in our experiments, the same precautions previously outlined have been implemented when handling and working with this bacterium. This strain was provided to us by Plant Env. Lab of the Department of Biochemistry and Biotechnology, University of Thessaly.

Pseudomonas Putida KT2440

The second chassis organism we introduced to our synthetic consortium was Pseudomonas Putida KT2440. This specific bacterium, known for its unique metabolic capabilities, is often described as “Generally Regarded as Safe”, or GRAS1. The GRAS status, determined by the FDA, is only awarded to chemicals or specific strains of microorganisms that are proven to be safe for ingestion2. In addition, it is a non-virulent strain3, minimizing its potential harm to humans. However, we remained committed to implementing safety measures within the lab, conducting the practices we explained earlier for the other bacteria. Its role in our consortium revolves around the degradation of the phenolic compounds found in the OMW while also utilizing this organic substrate-along with the FFAs E.coli BL21 produces-for producing PHAs.

We submitted a check-in form to the Safety and Security Committee, which was accepted on July 7, 2023.

The specific strain was kindly provided to us by Dr. Christos Batianis, Postdoctoral Researcher at the Bioprocess Engineering Group, Unit of Biomanufacturing and Digital Twins, Wageningen University & Research.

For some experiments, the utilization of hazardous substances or techniques was unavoidable. With our instructor’s guidance and training, our team members took diligent measures to adhere to all rules and guidelines; to prevent any harm to themselves or the environment.

-Olive Oil Mill Wastewater (OMW)

OMW is the by-product derived from olive oil processing, which we want to utilize for the cultivation of our synthetic consortium, to metabolize its organic substances and generate PHAs. OMW's main organic load consists of phenolic compounds such as hydroxytyrosol, protocatechuic, p-cumaric acid, vanillic and caffeic acid (4) and it is characterized by COD 30-320 g/L, BOD 35-132 g/L, and pH 2,2-5,9 4,5. The reason we wanted to experiment on OMW was to measure the efficient dilution for the survival of both our microorganisms.

We submitted a check-in form before conducting any experiments, and it was accepted by iGEM’s Safety and Security Committee on July 7, 2023,

OMW was provided to us by Mr. Spyroulis’ and Mr. Kantikos Olive oil mills.

-UV light

The utilization of UV light was essential for visualizing our electrophoresis results in agarose gels. All safety measures were taken to avoid exposure to UV light.

-Ethidium bromide

Ethidium Bromide was needed for the agarose gel visualization since it can bind to DNA. For the same reason this chemical is regarded as mutagen and is moderately toxic after acute exposure, team members followed all requirements to stay safe.

-Antibiotics

Kanamycin, Chloramphenicol, and Spectinomycin were used for transformants selection. While working with these antibiotics proper safety measures were followed

One of the primary objectives we aimed to accomplish was to guarantee the security of our system, ensuring that it would remain confined within our controlled environment, the bioreactor, and not be viable or sustainable outside of it. For additional information regarding the safety aspects of our design, please consult the 'Biosafety' section available on our Design page.

From the beginning of designing oPHAelia we strived to ensure safety, by talking to olive oil producers, experts, and studying relevant literature. This helped us ensure our procedure is safe and identify potential risks for the future, working on how in the future we will have to proceed to mitigate them; from collecting OMW from the olive oil mills to our biological system and dealing with the bioproducts generated in our facility. You can find more information about the safety of our proposed implementation which is explained in the relevant page (Implementation).