To prove the comprehensive validity of our proposal and contribute to the battle against eutrophication, there are three primary aspects to examine: the operation of freshwater monitoring; the development and testing of the floating platform; and a plant's response to the presence of microcystin by removing phosphorus from nutrient-rich waters. The design of the boat, its 3D printing, the installation of mobility electronics and sensors, the transmission of data to an application, and the operational testing of our boat have shown the effectiveness of the monitoring system. As far as CFW development is concerned, the platform was designed first, followed by mycelium cultivation and stabilization on the platform, and finally material testing. To demonstrate the biological system’s viability, in silico design and testing of a microcystin targeting aptamer for the simulation of a synthetic riboswitch that can bind MC-LR, partial characterization of the Pi Transporter expressing cassette under the regulation of the TetR protein, and testing of the plant's nutrient uptake capacity were conducted.

RC Boat

From the very beginning of our project, one goal we wanted to achieve was accurate, fast, and open access upload of pollution data on an operating water body. We achieved this through the creation of a Remote Control Boat (RC Boat). The RC Boat carries sensors related to the phenomenon of eutrophication, including a DO sensor, a pH sensor, and a GPS module. Here are all the steps needed to ensure the efficient function of our boat.

3D Design and Printing of the RC Boat

We designed our boat with the help of the software Fusion 360 by Autodesk. There were many things we needed to take into consideration. These included the buoyancy, the installation of all compartments for the boat, and the attachments of our sensors and Arduino. After extensive analysis and talks with experts, we began discussing design strategies. In the end, we split our boat into three parts: The main body, the cap, and the cylinders used for sensor induction. Because sustainability and circular economy are essential parts of our project, we wanted to use a material that does not have any negative footprints on the operating water body. Therefore, we decided to use Polylactic acid (PLA) as our material. As it is created from natural or recycled materials, PLA is embraced for its environmental friendliness, biodegradability, and many other characteristics. For further safety, we added layers of paint to our boat. This way, we minimize absorbency during our operations.

Sensors

The reason pH and Dissolved Oxygen sensors were chosen for our project is that, they are two of the most reliable and accurate indicators of eutrophication pollution inside a water body. Both sensors (pH and DO) were installed and programmed to the Arduino microcontroller. Installing the sensors to the Arduino was fairly easy as there is lots of documentation about it. You can learn more about calibrating the sensors on our software page

Having calibrated the sensors we made a test on saved lake Karla’s water and the results we got were very reasonable, knowing how eutrophicated the lake is.

• pH is basic at value 8.57, which can be characterised as low for a highly eutrophicated water body like lake Karla, but still indicates the eutrophication of the water body. (normal pH values for a lake range from 6 to 8)
• DO is very low at a price of 4.35 mg / L specifying in contrast with pH value that lake Karla is highly eutrophicated. (eutrophicated bodies DO, values range from 4 to 6 mg /L)

Figure 1. The test in water quality of Lake Karla’s water.

Figure 2. Both parts of the Arduino code and the results we got from the test

Real-world application and results

Web-application

So far, we have programmed the sensors to communicate with the arduino, the arduino to send the information of the sensors and the exact location of the information online, on cloud. Then online, the server would run the machine learning model, which would classify the places that have microcystins that our plants detect. The only part missing that would nicely fit in our problem and supplement our approach in solving eutrophication was the visualisation of the data captured. Through the application built, we provide an easy way to help people where exactly to place the CFWs, but also a useful tool that would raise awareness for the issue. The web-application built gets the data captured from the sensors and with the help of various packages and APIs under the Vue.js framework can visualise the data in the form of a heatmap. This heat map shows the areas of the eutrophicated points in the water body, with gradient colouring (from highly (red colour) to lowest (blue colour) intensity of eutrophication), and where the mycelium CFW should be deployed for optimal efficiency.

App's Output

RC Boat

The first real-world testing of our RC Boat took place on the river Pineios. During the procedure, we examined how our model behaves inside a water body. We got to see its balance when moving and turning. We also made a few tweaks in component placement on the inside of the boat. This way, we ensured optimal weight distribution and mobility. Furthermore, we adjusted the boat's shaft to guarantee its sturdiness and the model's reliability.

Figure 3.A top view of the RC Boat

Introduction

A fundamental aspect of this year's project was the platform where water bioremediation is achieved. One objective of ours was the use of sustainable materials for the construction of the wetland. Therefore, we decided to use mycelium, an eco-friendly material without negative footprints on the operating waterbody. For all this to be possible, we had to ensure all safety precautions and functionalities of our blueprint. These include an early design of the volume where our platform will grow, the mycelium growth process, and its implementation. We accomplished these goals through the following steps:

Hardware

3D design and printing of mold for wetland

To create the shape of our wetland, we initially had to construct a mold design where our mycelium structure would grow. Therefore we created a prototype sketch using the software Fusion 360 by Autodesk. After creating wetlands of various shapes and sizes, we created a circular model of a 40cm diameter consisting of 37 holes for our plants.

Figure 4. 3D Design of mold

Growth of mycelium

After creating the mold where the platform is made, we completed the following steps:

1. Preparation of the biomaterial. The material is in a sterile bowl. We then combined it with flour and a sculpting mix. Later on, we made sure our material did not have any lumps. This procedure requires wearing gloves.
2. We then sprayed the mold with an ethanol solute while ensuring proper sterilisation. Following this, we added the substrate to the entire surface of the mold.
3. Once the mold is filled, we cover the area with plastic foil while creating small holes of about 3cm. It was necessary for the product to have sufficient air at all times.
4. We let it grow for 3- 5 days at 21° - 24°C. It should be gently removed from the mold.
5. Finally we placed the product in the oven at +/- 40° C with the door a bit open so the moisture can escape for 3-4 hours. Afterwards, we baked it at 80°C for 2 hours to provide more stability to our model.

Real-world application and results

After taking everything into consideration and completing all previously mentioned steps, it was time to implement our platform in real-world conditions. We tested our Constructed Floating Wetland inside a water tank. There we could accurately test our platform's floatation and overall behavior inside the water body.

In the end, our platform remained stable and able to handle all factors it might face in real-world situations (e.g. wind, high water flow, alternating weather conditions). The circumstances should be no different once the plants are placed. The platform's mechanical specifications can withstand the additional weight without further complications.

The part of our project’s concept we wanted to prove in the Wet Lab is that the biological system that we designed could make a plant respond to the presence of microcystin with phosphorus uptake from nutrient rich waters, and help combat the phenomenon of eutrophication. It is very important for us to highlight the distinction between the experimental design that we had planned, and how much we actually achieved in the 2 1/2 months that we spent in the lab.

Microcystin Detection

Once the platform equipped with the engineered plants is placed in the water, expression of the nutrient removal system will take place upon detection of the eutrophication biomarker, microcystin-LR. This is done with the synthetic riboswitch that we designed (Engineering success). A microcystin targeting aptamer was combined with the thiamine pyrophosphate (TPP) riboswitch, and the in silico screening of possible sequence candidates resulted in a library of riboswitches that can, theoretically, uphold ligand binding and control mRNA degradation of the repressor module.

Figure 5. Complex of microcystin bound to the new riboswitch.

Figure 6. Representation of the Detection Module in the absence (left) and presence (right) of microcystin-LR.

To prove that TetR expression can indeed be regulated with any kind of riboswitch, we planned to test our system with the unmodified TPP riboswitch. To do that, we needed TPP-auxotrophic plants, which unfortunately we could not obtain. We tried to use another riboswitch; a theophylline aptazyme, but we were not able to assemble this part into our constructs.
Aside from riboswitch control, we found that the TetR protein could be successfully expressed in plant systems both on its own (Figure 7), and combined with the KRAB protein (Figure 8) (see the Experiments page).

Figure 7. Agroinfiltration experiment: pNOS-Venus-TetR-tNOS.

Figure 8. Agroinfiltration experiment: pNOS-Venus-TetR-KRAB-tNOS.

PHT1 expression

The design of the second module is aimed at placing the PHT1 expressing cassette under the regulation of the TetR protein. Our experiments went as far as confirming the assembly of the DNA parts that constitute the second module. However, it was our intention to perform transient gene expression in N.benthamiana leaves with agroinfiltration, to also confirm the successful localization of the PHT1 proteins in the cell’s membrane. Moreover, the next step would be the “hairy root” transformation of L. japonicus to study the response of our system to the presence of an actual activator. This would be either thiamine pyrophosphate or theophylline, as mentioned before.

Figure 9. Representation of the Nutrient Uptake Module in the presence(left) and absence(right) of TetR protein.

Increased phosphorus uptake

Had our experiments arrived at the stage of the hairy root transformation, we would need a method to test the nutrient absorbing abilities of the plant. The selection and development of this method was greatly facilitated by team Manchester. In the context of our partnership, iGEM Manchester shared with us the phosphorus measuring method that they would use for their own project. 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).
We managed to perform the calibration step with K₂PO₄ solutions and we also measured the concentration of phosphorus from water that we collected from the local river and lake. The measurements were similar to the ones reported in literature. Thus, we would be able to set-up an experiment with a hydroponic culture of the modified L. japonicus. The water would be rich in phosphorus, and after a week, we would use the phosphorus measuring method to evaluate our system’s functionality.

To demonstrate the real potential and viability of our concept, we went far beyond imagining hypotheses and conducting scientific literature research to support them. Our vessel's design, 3D printing, installation of mobility electronics and sensors, data transmission to an application, and operational testing have shown the monitoring system's effectiveness. Concerning the development of CFW, the platform was designed initially, followed by mycelium cultivation and material testing. To demonstrate the viability of the biological system, the in silico design and testing of microcystin targeting aptamer for the simulation of a synthetic riboswitch that can bind MC-LR were conducted, as well as the proof of the functionality of our basic parts for the expression of Pi Transporter under the control of TetR protein.