Project 2021

Team Aboa 2021

The following description is from our wiki page:

Our home town Turku is located on the coast of the Baltic Sea. The Baltic Sea is a young and ecologically very sensitive sea area, which is under threat by human activity. It’s a very important area to local residents and for many, including us, it offers experiences and opportunities for relaxation. It was clear from the beginning that we wanted to come up with a solution to protect the sensitive and unique ecosystem of the Baltic Sea.

The picture of Aboa’s team members kayaking.

The Problem

The Baltic Sea is one of the most polluted seas in the world. One of the biggest problems is pharmaceutical waste - it has been accumulated in the Baltic Sea already for decades. (The John Nurminen Foundation.) In addition to the well-known pollutant agents, antibiotics and hormones, anti-inflammatory drugs are also a huge problem (UNESCO & HELCOM, 2017). Especially diclofenac, a non-steroidal anti-inflammatory drug (NSAID) which is used mainly in topical pain-relieving gels, has been recently determined to be a severe threat to the delicate ecosystem of the Baltic Sea (Ek Henning et al., 2020). It, among other things, accumulates in fishes and mussels and damages their renal and gastrointestinal tissues (Lonappan et al., 2016; Vieno & Sillanpää, 2014).

When we did more research and interviewed our local wastewater experts, it became clear that diclofenac is a huge problem. As you can see from the picture below (Figure 1), the situation in our local wastewater treatment plant (WWTP) is quite bad.

A picture of the sea in the front of which there are two boxes with the following texts: “90 kg of diclofenac end up in the Baltic Sea every year” and “27 % of diclofenac can be removed in the wastewater treatment process.
Figure 1. The amount of diclofenac ending up in the Baltic sea from our local WWTP and the current removal efficiency of diclofenac in our local WWTP.

After doing even more research on diclofenac, we noticed that something is clearly wrong regarding its removal process. Considerably more diclofenac ends up in the Baltic Sea when compared to other often used pharmaceuticals. In 2014, 77 250 kg of diclofenac was used in Finland and as much as 845 kg of it ended up in the bodies of water (Figure 1). That is over one percent of the whole amount. When it comes to more commonly used drugs, ibuprofen and paracetamol, the percentages are 0,1 % and 0,3 %, accordingly.

Every year hundreds of kilograms of pharmaceuticals end up in bodies of water in Finland. In the picture, there is diclofenac, carbamazepine, ibuprofen and paracetamol. 77 250 kg of diclofenac was used in Finland in 2014 and 845 kg of it ended up in bodies of water. For carbamazepine, numbers were 3 321 kg and 190 kg, in the case of ibuprofen 117 825 kg and 130 kg and for paracetamol 194 685 kg and 630 kg, accordingly.
Figure 2. The amounts of certain pharmaceuticals ending up in the Baltic Sea. (Modified from source: SYKE - Finnish Environment Institute).

Read more about pharmaceutical waste here.

Why did we choose this topic?

In the Turku region there are many pharmaceutical and diagnostics companies as well as a strong expertise cluster in health research by universities, so we believe that a solution to the problem can be provided. We believe that private individuals can also make a difference for the wellbeing of the sea and that’s how our iGEM project began.

It was obvious to our team from the very beginning that we wanted to do something related to the bad condition of the Baltic Sea. We searched for different pollutants from microplastics and cosmetics to dyes and pharmaceutical waste. Since we had team members studying biomedicine and chemistry of drug development, we ended up doing our project on the topic of pharmaceutical waste.

After we had narrowed our topic down to pharmaceutical waste, we started to gather information about it. It quickly became clear that the biggest and the most problematic pharmaceutical pollutants of the Baltic Sea were antibiotics, hormones and anti-inflammatory drugs, such as diclofenac (UNESCO & HELCOM, 2017). The amount of diclofenac in the bodies of water in Finland surprised us and we wanted to get more information about it.

When searching for more information about diclofenac, we found three previous iGEM projects from the same topic. The German teams Darmstadt (https://2020.igem.org/Team:TU_Darmstadt), Kaiserslautern (https://2020.igem.org/Team:TU_Kaiserslautern) and Stuttgart (https://2020.igem.org/Team:Stuttgart) had carried out their projects about diclofenac in the bodies of water in the iGEM competition 2020. We familiarized ourselves with their projects and contacted them for getting more information. We were really inspired by their projects and decided to execute our own project on diclofenac!

We searched for different bacterial laccases and ended up with three different laccases: CotA from Bacillus subtilis, CueO from Escherichia coli and Yak from Yersinia enterocolitica subsp. palearctica strain 7. Of those three laccases, CotA and Yak have been proved to be able to detoxify diclofenac and thus they were obvious options for our project (Chen et al., 2020; Arregui et al. 2019). However, Yak hasn’t been used in the iGEM projects before. CueO, in turn, was one of our novelty factors since, to our knowledge, it hasn’t been studied in degrading diclofenac before. Read more about our laccases and the design of our project here.


Our Solution

We decided to aid the situation of the Baltic Sea by contributing to the development of a microbial wastewater purification system. Our aim was to compare the activities of three laccases; CotA, CueO and Yak. The best one of them would have been introduced into our end host Synechocystis sp. PCC 6803. Our ultimate goal was to implement our solution to our local WWTP in future. Cyanobacteria would have produced and secreted laccase in a closed photobioreactor system, from which the laccases would have flown through a filter first to a “kill tank” and then into the wastewater. With this type of system, we would prevent any GMOs from spreading to surroundings. Read more about our proposed implementation here.

Since pharmaceutical pollutants in the bodies of water are a remarkable and current problem, we believe that our project is significant and important in contributing to the problem and raising awareness of it. Our project is a useful application of synthetic biology since there is an urgent need for better solutions to degrade pharmaceutical waste and with the help of synthetic biology, we can do it sustainably. Our previously mentioned end host, cyanobacteria, is able to stay alive only with light, carbon dioxide and water due to its photoautotrophic nature.

A graphical illustration of our project. In the first step, there are three plasmids of which each contains one laccase gene name. From plasmids arrows point to the bacteria, under which there is a text “E. coli BL21(DE3)”. Arrows point from the bacteria to structures of laccases and further to one 96 well-plate. From this well-plate one arrow is pointing to a graph and further to a petri dish containing cyanobacteria. Finally, an arrow is pointing to a picture in which there are green tubes indicating a photobioreactor as well as gray pipelines and a tank indicating a wastewater treatment plant.
Figure 3. Overview of our project. First, we designed plasmids for the overexpression of the laccase enzymes CotA from Bacillus subtilis, CueO from Escherichia coli and Yak from Yersinia enterocolitica (1). Then we transformed the plasmids into E. coli BL21(DE3) cells (2) and purified the produced laccases (3). We measured laccase activities with ABTS and syringaldazine assays (4). Our aim was to compare the laccase activities at conditions simulating our local wastewater treatment plant (5), and express the most promising laccase in the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (6). These laccase-producing cyanobacteria could then finally be implemented as a part of a wastewater treatment plant in the form of a photobioreactor (7).

Our goals and how to achieve them:

The aim of our project was to aid the situation of Baltic Sea by developing a system of engineered microbes that could detoxify diclofenac to less harmful compounds. That system could be implemented to the wastewater treatment processes in the future. We tried to achieve this aim by researching three different laccases and comparing their efficacies. The laccases that we chose were CotA from Bacillus subtilis, CueO from Escherichia coli and Yak from Yersinia enterocolitica.

Another remarkable aim of our project was to settle the iGEM culture and the annual participation in the competition in Turku. This year has been extremely hard for our team and the main reason is that it has been difficult to get support for our project due to the unknown nature of iGEM competition here in Turku and even in Finland. Because of that, we decided to try to root this competition in Turku and allow it to become an annual tradition. We tried to achieve this aim by distributing information about iGEM as well as visiting schools and fairs. In addition, we got the opportunity to be interviewed by many local newspapers. Read more about our efforts from here. On top of all of this, we really put effort into trying to enable future Aboa teams to get study credits for their project at our university.


Sustainable Development Goals:

Our project is also tackling worldwide problems when looking from the perspective of Sustainable Development Goals (SDGs) (United Nations). Our project addresses the following goals:

A picture about four Sustainable Development Goals to which our project is related.

3. Good health and well-being: Our project contributes to this goal by reducing the diseases that are caused by the pollution of seas and other water ecosystems.
6. Clean water and sanitation: Our project contributes to this goal by trying to improve the quality of water. In Target 6.3 it is mentioned that one of the aims in this goal is to reduce pollution and minimizing release of hazardous compounds in water as well as to halve the amount of untreated wastewater. Thus, our project contributes to this goal especially by purifying water from pharmaceuticals.
12. Responsible consumption and production: Our project contributes to this goal by using photoautotrophic cyanobacteria as a host for our final solution. Cyanobacteria are a sustainable option as they get their energy from light, carbon dioxide and water through photosynthesis.
13. Climate action: Our project contributes to this goal by providing a sustainable cyanobacteria-based solution. As mentioned earlier, they are photoautotrophs, thus offering an environmentally friendly production method.
14. Life below water: Our project contributes to this goal by decreasing the amount of diclofenac in seas. This drug substance has been shown to harm the organisms of the water ecosystem, especially to fish and mussels.
15. Life on land: Our project contributes to this goal by minimizing possible harm to organisms living on land. For example, birds can be exposed to the harmful effects of diclofenac when eating fish.


Covid-19’s impact on our project:

Fortunately, our team was able to design and execute a project in a wet lab since the Covid-19 situation was much better here in Finland during the summer of 2021. However, Covid-19 did affect us earlier in the spring when we were about to start our project. Because of general movement restrictions at the time, we weren’t able to see each other in person for many months and this slowed down the process of grouping in our team.

Grouping is an essential part of becoming an approved member of a team. Grouping happens when people interact with each other sharing and receiving verbal and non-verbal signs and messages. When interpreting these straight-forward or subtle signs, people start to understand the dynamics within the group. By doing this, people will gain important knowledge on things such as who to follow when making decisions and who to turn to when a group needs a quick relief from a tense moment.

In order for a person to be able to be the best version of oneself in a competitive group like an iGEM team, one should be able to be who they are free of judgement and doubt. Getting to know a person simply takes time and limiting many of the non-verbal signs like posture and direction of your body, inevitably slows down the process of learning to interpret one another in a group setting. Not getting to know each other in person also slowed down the process of daring to speak out your mind in the beginning. This is an extremely essential part of e.g. the ideation phase.

Luckily, we were able to meet each other multiple times in person during the summer, and our small team got to eventually benefit from the best expertise and skill set of each member of the team. We also managed to have a lot of fun during different phases and points of the project and to be honest, what can you say, diamonds are formed under pressure.


References

  • Arregui, L., Ayala, M., Gómez-Gil, X., Gutiérrez-Soto, G., Hernández-Luna, C. E., de los Santos, M. H., Levin, L., Rojo-Domínguez, A., Romero-Martínez, D., Saparrat, M. C. N., Trujillo-Roldán, M. A., Valdez-Cruz, N. A. (2019) Laccases: structure, function, and potential application in water bioremediation. Microbial Cell Factories, 18(1), 200. https://doi.org/10.1186/s12934-019-1248-0.
  • Chen, L., Li, Y., Lin, L., Tian, X., Cui, H., Zhao, F. (2020). Degradation of diclofenac by B. subtilis through a cytochrome P450-dependent pathway. Environmental Technology & Innovation, 20, 101160. https://doi.org/10.1016/j.eti.2020.101160.
  • Ek Henning, H., Putna-Nimane, I., Kalinowski, R., Perkola, N., Bogusz, A., Kublina, A., Haiba, E., Barda, I., Karkovska, I., Schütz, J., Mehtonen, J., Siimes, K., Nyhlén, K., Dzintare, L., Äystö, L., Sinics, L., Laht, M., Lehtonen, M., Stapf, M., Stridl, P., Poikäne, R., Hoppe, S., Lehtinen, T., Kõrgma, V., Junttila, V., Leisk, Ü. 2020. Pharmaceuticals in the Baltic Sea Region - emissions, consumption and environmental risks. Report no. 2020:28, Länsstyrelsen Östergötland, Linköping. Available at: https://www.lansstyrelsen.se/4.f2dbbcc175974692d268b9.html.
  • Lonappan, L., Kaur Brar, S., Kumar Das, R., Verma, M., Surampalli, R. 2016. Diclofenac and its transformation products: ENvironmental occurrence and toxicity - A review. Environmental International, volume 96. Available at: https://doi.org/10.1016/j.envint.2016.09.014
  • The John Nurminen Foundation: Information about The Baltic Sea. Retrieved August 16, 2021 from https://johnnurmisensaatio.fi/en/baltic-sea-protection/information-about-the-baltic-sea/.
  • UNESCO and HELCOM. 2017. Pharmaceuticals in the aquatic environment of the Baltic Sea region - A status report. UNESCO Emerging Pollutants in Water Series - No. 1, UNESCO Publishing, Paris. Available at: https://helcom.fi/media/publications/BSEP149.pdf.
  • United Nations: Department of Economic and Social Affairs. The 17 goals - Sustainable Development. Retrieved October 13, 2021 from https://sdgs.un.org/goals.
  • Vieno, N., Sillanpää, M. 2014. Fate of diclofenac in municipal wastewater treatment plant - A review. Environmental International, volume 69. Available at: https://doi.org/10.1016/j.envint.2014.03.021.