Safety
Safety
The use of such advanced scientific tools must be preceded by a risk assessment and careful planning of experiments to ensure that all procedures are conducted with appropriate safety measures in place. Our team, in collaboration with our educational supervisors and advisors, made every effort to ensure that the entire project was carried out as safely as possible.
Once we selected the topic for our project, we began considering how to ensure safety, especially while working with Prymnesium parvum, which we detect using our tool called PrymDetect. This algae is harmful to gill-breathing organisms and can lead to ecological disasters, which is why preventing contamination outside the lab was our top priority.
We maintained continuous cultures of algae and isolated DNA from them to test the tools we developed. This allowed us to evaluate the effectiveness of PrymDetect and the approximate detection levels regardless of the season. To reduce the need for fresh algal cultures, we regularly performed PCRs to amplify the target DNA fragments to the necessary quantities for our experiments. To ensure the workflow of the project wasn't disrupted, we needed algal cultures as a backup source of DNA for PCRs. This way, if the PCRs failed and we lost our original template, we'd still have a reliable backup to continue our work.
In the future, algal cultures could be replaced with DNA produced from a plasmid containing the Prymnesium parvum DNA fragment needed for our tests, which can be multiplied in bacteria. We sequenced this fragment to make it possible to clone it into a plasmid, leaving this as a foundation for future iGEM teams interested in working with Prymnesium parvum.
This was the most challenging safety issue in our project. Firstly, we needed to understand how the toxins released by Prymnesium parvum work. We discovered that they cause the death of gill-breathing organisms by interacting with the membranes of gill cells [1]. These membranes begin to develop holes, causing ion concentrations to equalise on both sides. Consequently, the gills lose their ability to absorb oxygen from water.
Understanding this process allowed us to assess the risks associated with working with these organisms. The main risk was accidentally releasing the algae into the environment, for example, through sewage. Therefore, every water sample taken from reservoirs that could contain algae, as well as cultures and containers used to store the algae, were killed using sodium dichloroisocyanurate. According to the literature, this substance is an effective biocide against algae [2,3].
It is also worth noting that the presence of this algae has been detected in many water bodies and rivers across Poland, and from such sources we obtained material to establish our cultures.
It's worth mentioning that before starting our work with golden algae, we submitted the iGEM Check-In form, which was approved by the safety committee. As a new team, we wanted to make sure our work adhered to iGEM's safety guidelines from the outset.
We also worked with Escherichia coli strains. The strains we used included DH5α, Rosetta™ 2(DE3)pLysS, TOP10, and NEB® 5-alpha Competent, all of which are classified as Risk Group 1 organisms (non-pathogenic and low risk to humans).
Despite this, several risks must be considered when working with these strains, such as the spread of antibiotic-resistant genes to other bacteria, unintended overexpression of proteins, allergic reactions in some individuals or accidental release of the GMOs into the environment. That’s why we inactivate them after use and follow Good Microbiological Practice (GMP) guidelines.
Before starting work in the laboratory, each team member underwent training on safety protocols, Good Microbiological Practice (GMP), waste disposal, and equipment handling. Some of us also received training on operating the benchtop autoclave and chemical fume hood. We worked in well-equipped laboratories that included first aid kits and eye wash stations. To ensure our safety, we used personal protective equipment such as lab coats, gloves, and safety glasses. Additionally, while working with the Cas13 protein, it was necessary to use a sonicator. Due to the emitted ultrasound, we used noise-cancelling headphones to avoid hearing damage and refrained from staying in the room during sonication. We also adhere to rigorous waste management and sterilisation protocols, and follow strict procedures.
Thanks to our supervisors and other lab workers we gained knowledge and good habits that not only we will benefit from but also our colleagues and the whole society and environment as we will think broadly about safety issues.
While working with parts from the iGEM distribution kit, we dealt with bacterial cultures that were resistant to various antibiotics. To minimise the risk of accidentally using the wrong antibiotic, we implemented our own colour-coding system for labelling plates, Eppendorfs, and flasks containing bacterial cultures, according to the antibiotic colour code we established. This helped reduce the risk of developing antibiotic resistance in the bacteria.
We also meticulously labelled and stored samples to prevent mix-ups. The most effective way we documented and catalogued samples was in the laboratory where we cultured algae. Our naming conventions included the sample's origin and passage numbers, allowing us to monitor changes in cultures after subsequent passages and assess the state of the cultures.
The initial ecological disaster in the Oder River in 2022 was surrounded by a lot of misinformation. In conversations with the community, we noticed that there is still widespread confusion and mistrust regarding the actual cause of the disaster and the safety of the affected waters for human health. Despite the fact that over two years have passed since the initial disaster, the topic of algae remains shrouded in mystery for the public. Therefore, one of our goals was to educate people on this subject. We originally planned to conduct a survey to assess public awareness of golden algae, but we abandoned this idea due to the risk of causing public panic. Instead, we decided to participate in a radio broadcast, where, along with our scientific supervisor, we aimed to explain the mechanisms of algal blooms and answer the most frequently asked questions based on current scientific knowledge. We believe that explaining to people this mechanism in an easy way is the best solution for raising social awareness. We also recognize that the widespread availability of our test could lead to unnecessary public panic; therefore, before any potential market introduction, a large-scale educational campaign should be conducted, and each test should include instructions explaining the actual scale of risk associated with detecting the algae.
We are confident in our abilities and committed to maintaining the highest standards of safety throughout our work. If any doubts arise, we can always consult our supervisors, who are readily available to assist us. Additionally, we prioritise the workload and well-being of our team members to ensure they are not overworked, thereby reducing the likelihood of accidents.
[1] https://pubs.nmsu.edu/_circulars/CR647.pdf
[2] White GC. Handbook of Chlorination and Alternative Disinfectants. 4. Wiley; 1998
[3] Seo D, Lee YH, Jo J. Humidifier disinfectant, sodium dichloroisocyanurate (NaDCC): assessment of respiratory effects to protect workers' health [published correction appears in Sci Rep. 2024 Apr 19;14(1):9023. doi: 10.1038/s41598-024-59792-z]. Sci Rep. 2021;11(1):15681. Published 2021 Aug 3. doi:10.1038/s41598-021-95148-7
