We, iGEM Thessaly 2022, recognize the importance of cooperation with another team throughout the year and consider it an essential factor for the project development. Therefore it was necessary to reach out to another team who shared similar concerns and goals and worked together as partners to achieve a more holistic view on environmental impact. During the competition, we had the opportunity to engage with team iGEM Manchester and form a partnership with them.

Our cooperation with iGEM Manchester helped both projects in numerous ways. The Wet Lab departments established a simple method for phosphorus quantification from environmental samples, which will confirm our plant's phosphorus removal capabilities and supply iGEM Manchester with reliable data for their model. In Dry Lab, we expanded our horizons by analysing ways the project could be used across various water bodies.Furthermore, through our mutual activities, we gathered information regarding GMO legislation, an integral part of both projects' implementation.

Since this year, our project centres on the phenomenon of anthropogenic eutrophication, one focal point was finding a team dedicated to resolving challenges directly related to the phenomenon. Through overviews and short descriptions of each team's project we discussed strategies for collaborations with other teams. After a while, we stumbled across team iGEM Manchester and their project 'BloomAid.'

Through the project 'BloomAid' team Manchester aims to tackle wastewater mismanagement and the need for biofuel production. Their strategy revolves around microalgal production and use. They can achieve this through developing an engineered bacterium that assists in microalgal growth and the accumulation of lipids. This will subsequently be converted into biofuels. Microalgal growth is a direct effect of eutrophication pollution. Their strategy consists of the implementation of a co-cultivation system between engineered bacteria and microalgae in an enclosed bioreactor, that would enable the usage of wastewater as a growth medium and the enhancement of the yield of the lipid precursor stage towards biofuels. The system will function on two modes: the first one is based on the engineered bacteria using the organic waste as a substrate to produce auxin, an algal growth factor, that stimulates the rate of growth of the microalgae. Once sufficient algal biomass has been produced, the engineered bacteria can be triggered through a light induced genetic switch mechanism based on a specific wavelength to move towards a phosphate-accumulating state, that would generate the depletion of the high concentrations of phosphates from the wastewater medium. This mechanism will induce the second mode which encloses the subsequent phosphate starvation in the microalgae, allowing for the enhanced production of monounsaturated fatty acids which can be harvested and used to produce a greater yield of biofuels.

Therefore we immediately thought of different ways of approaching and connecting our projects. Because our teams share the common theme of eutrophication, there are many prospects in which we could help each other. From the beginning, it was evident we had similar ways of approaching solutions in our projects. We both focus on promoting sustainability, circular economy, and green energy., whether it is through biofuel production or using biodegradable and eco-friendly materials. Therefore it was reasonable for us to join efforts with iGEM Manchester for 2022.

Brainstorming

Even though we both shared a similar vision in combating eutrophication, our teams had followed vastly dissimilar paths in our Wet Labs. We used plants with the intention of phyto-remediating eutrophic lakes in situ, while team Manchester used a bacteria-algae consortium to clean up phosphorus from wastewater. It seemed very unlikely that we would find any common ground to work on. However, after several meetings, we began to search for a method of measuring Phosphate levels in various media that would provide transferable results between our laboratories. This is what, how, and why we did it.

Plan of Action

A significant gap in our experimental design was the lack of a final step that would confirm the phosphorus removal capacity of our engineered plant. Once we had the plant ready, we would need a way to test that. Team Manchester, though, had figured this out for their own design and suggested that we follow the same procedure. It is a commonly used method for the colorimetric determination of phosphorus and it is based on the reagents ammonium heptamolybdate and ammonium metavanadate (or vanadate-molybdate in short). Ammonium molybdate reacts under acid conditions to form molybdophosphoric acid. In the presence of vanadium, yellow vanadomolybdo-phosphoric acid is formed, with the intensity of the yellow colour being proportional to phosphate concentration. Absorbance is measured at 470 nm. Water from all sorts of samples can be used.

This is a very simple method. Initially, a standard curve was calculated for calibration, with 5 different concentrations of KH2PO4. Then, we prepared the phosphorus-rich water samples reactions by adding vanadate-molybdate, shaking thoroughly and waiting 10 minutes for the colour to develop fully. We measured the absorbance at 470 nm and plotted the results.

Figure 1.Different concentrations of K₂HPO₄, combined with the molybdovanadate reagent during the 10 minute waiting step.

Figure 2. The absorbance is related to phosphorus concentration at a ratio of [P]/Abs = 7,2158

Despite getting this method ready for phosphorus measurements, we did not reach the point of plant root transformation due to time limitations. We had no sample to test. However, we could still take some measurements from local water bodies that suffered from eutrophication, like river Pinios and lake Karla. This way, we simultaneously tested that the method worked as expected, and provided our friends from Manchester with quality data that they could use in their model. This helped them investigate the possibility of implementing their project in lake ecosystems.

Brainstorming

Our Dry Lab departments spent a couple of meetings discussing the key similarities and differences in our projects and approaches. Even though both projects focus on the phenomenon of eutrophication, we differ in our approaches and implementations. For starters, the water bodies we most operate in are lakes and reservoirs, while iGEM Manchester focuses its efforts on rivers. Additionally, our subdivision's primary goal is to predict the presence of toxins inside the water due to pollution. Meanwhile, Manchester's Dry Lab emphasizes on a specific aspect of the phenomenon, the presence of microalgae. Afterwards, the collection of microalgae leads to biofuel production. Through our conversations, we agreed to focus on an area where we could provide an authentic solution. During our research, one thing that stood out was the lack of available and up-to-date data on eutrophication. That being the case, we collectively decided to implement a monitoring system that can operate in various water bodies. Our end goal is to develop a comprehensive and accurate framework that provides an instant update on the ecological status of the operating water body.

Plan of Action

Our strategy was both unique and quite demanding. We decided to create a database that will include various parameters directly related to the phenomenon of eutrophication. Our work consists of two parts, one operated by iGEM Thessaly and one by iGEM Manchester. When it comes to iGEM Thessaly, we used sensors such as pH, Dissolved Oxygen (DO). This way we can accurately predict, through machine learning, the existence of toxins (Microcystins L-R) inside the water body. In the meantime, team iGEM Manchester is designing a model that operates using a variety of other indicators of eutrophication. These include Total Phosphorus (TP), Total Nitrogen (TN), and Phosphate. In addition, they also evaluate how these indicators are distributed around a point that shows high levels of eutrophication. Their goal is the effective modelling of microalgal growth for later biofuel production.

We achieved database development with the use of excel and CSV files and with the help of Jupyter notebook. These files consisted of a large number of values from many eutrophication-related parameters. These include pH, Total Phosphorus(TP), Total Nitrogen(TN), Dissolved Oxygen (DO), Temperature, Depth, Opacity, Chlorophyll-a concentration. Through the data rows we gathered, our team was able to create a machine learning algorithm, which uses a boolean data type, and can predict whether our operating water body has toxins or not. We sent these values to iGEM Manchester to help them with their project. Their Dry Lab can precisely determine the growth rate of microalgae by measuring TP, TN, and especially chlorophyll-a concentrations, which are among the most significant indicators of microalgal development.

The model developed by iGEM Manchester can analyze the distribution of many nutrients, like Phosphorus, inside the operating water body. It also uses some of the parameters mentioned before, such as DO, Chlorophyll-a, Temperature, and TP. This way, we can also determine the amount of pollution caused by eutrophication in the water. As a result, this model helps our team dictate the optimal location for placing our Constructed Floating Wetlands by indicating the places most affected by pollution. Lastly, the model developed by iGEM Manchester can help our project expand our work on additional waterbodies. Through their help, we can more accurately analyze pollution in rivers.

Lastly, both the database and the model provide both projects with an extra step of revision and verification. For every calculation or analysis we make, we can use the software created by the other teams to test our results and their legitimacy. The database and the model helped both teams gain valuable information on accurately analyzing eutrophication pollution on many different water bodies. As a result, both projects are enhanced. The systems developed by our teams have helped our Dry Lab analyze the existence of microcystins with precision. Meanwhile, iGEM Manchester's Dry Lab can measure microalgal growth rate in extensive detail.

Brainstorming

Upon receiving feedback, our teams collectively concluded that one supplemental goal of ours should be to raise awareness about the phenomenon of eutrophication and the scientific field of synthetic biology. Topics that, in general, remain relatively unfamiliar to the public. After several meetings between our Human Practices departments, we collectively decided that our focus should not only be on the promotion of our projects and the problems we are trying to solve but also on informing people about synthetic biology. Furthermore, in order to successfully implement our initiatives, we wanted to discuss and learn more about universal GMO regulations that exist in many countries, as well as educate and inform the general public on the subject.

Plan of Action

Podcasts

Initially, we thought it would be a good idea to record a series of podcasts. Expressing our views through a podcast was, according to our teams, an effective way to reach a broad audience considering the number and age groups that choose these platforms to educate and entertain themselves. In these podcasts, we discussed various topics ranging from a short introduction to our projects to the importance of synthetic biology. We also addressed both the advantages and limitations of synthetic biology and why we believe public opinion still has a predominantly negative stance towards it. Another point of interest was the concept of the iGEM competition and why we find it so intriguing. Since our project uses genetically engineered plants, one more fundamental aspect of our project was understanding GMO legislation from across the globe. This way, we are informed of all procedures and actions we need to take. It also ensures the safe and efficient application of our project. This led us to the organisation of a workshop with the hopes of raising awareness and educating the public on legislative issues concerning GMOs.

Workshop

GMO use brings about many complications concerning their application and the regulations around them. Therefore, we decided to organize an open-access virtual symposium on GMO legislation from around the world. The workshop was conducted alongside iGEM Manchester and in collaboration with iGEM Bath, and iGEM HKU. The topic revolved around worldwide GMO legislation with an emphasis on the challenges we might face when trying to implement our projects in alignment with our country's legislation. As our teams all come from different locations worldwide, it was essential to mention how different national administrations deal with GMO production and utilisation. This was important to our teams and the audience members who were present since we not only learned about the different legislations worldwide but also gave an idea about the countries we could implement our projects. We also invited a number of experts in order to voice their opinions and give us feedback on our ideas. All of this led to a very productive and informative discussion on the subject.

All departments: Wet Lab, Dry Lab, and Human Practices made significant efforts through our conversations and collective work. During the workshop we co-organized, both teams collected valuable information on GMO rules applied in many countries, which aided the implementation of the projects. Furthermore, our wet lab subdivisions helped one another out. Through the executed colorimetric method, we helped team iGEM Manchester find out their project's eligibility in lake ecosystems, and they were able to help us find a way to validate that our engineered plant could remove phosphorus. Last but not least, both teams established more effective eutrophication monitoring systems using a database and model developed by our Dry Lab departments, respectively. We are pleased and appreciative to have cooperated with the Manchester team and formed a partnership with them!