We decided to gather information on the problem of "paint conversion," which would be a problem for social implementation on ships in the project. We thought that the only way to prevent the attachment of sessile organisms on a moving ship would be to use a paint that contains cWA and that would slowly dissolve in seawater. Therefore, we had a meeting with Fujitsubo Guard Corporation, a company that research on currently method for anti-adhesion paints.

What was clear as a result of the discussions was the issue of intellectual property. Many of the ship-bottom paints recently used are patented by paint companies, and in order to actually create paints using cWA, they have no choice but to collaborate with such paint-related companies. As an opportunity to get involved with such paint companies, he told us about the Variship 2023 event, which will be described later in this report. In the meeting, he also introduced some of the research that Fujitsubo Guard has been involved in and explained how they have been trying to use adhesion inhibitors in ways other than paint. For example, they have been trying to mix them into certain porous materials.

Through the meeting with Fujitsubo Guard, we were able to learn about intellectual property issues that arise in social implementation. In addition, we got an opportunity to consider where the project could be applied and where human practice could go further through learning about approaches to social implementation other than paint. It also became a trigger for us to determine the need for a more detailed understanding of the physical properties of cWA when discussing the possibility of using it as a paint in the future with companies. It also provided an opportunity for further Human Practice in Variship so this Human Practice was very meaningful for us.

During our research on sessile organism attachment inhibition, we wondered how the effect of attachment inhibition was measured. We also strongly felt necessity to hear from those who are conducting such research and those who have a vested interest in the issue. We especially wanted to ask how our approach appeared to those conducting similar research and what problems they perceived.

Then, we interviewed Cecil Research Inc. Cecil Research Inc. is the only company in the world that specializes in the issue of sessile organisms. They have conducted many joint research projects with university researchers, and we thought we would be able to get a fair and honest opinion about our project.

At our first meeting, we explained our project and received a variety of opinions. One of the most important points raised was that we cannot claim that the fact that a substance is derived from a living organism does not necessarily mean that it does not have negative effects on all living organisms. To claim that a substance is truly good for the environment, he told us that it is necessary to conduct toxicity tests on many kinds of living organisms inhabiting the ocean. On the other hand, he said that is not very realistic and that we should rather promote the fact that it has no adverse effects on sessile organisms. The adhesion inhibitors that have been used to date clearly affect the survival of sessile organisms and have a negative impact on the marine ecosystem via sessile organisms. Our project was highly praised as a possible breakthrough solution to this problem. In terms of social implementation, he also suggested that it is not realistic to start with ships and power plants, but that we should take a gradual, step-by-step approach, starting with niche applications.

Then we had a meeting again during the iGEM activity period. At that time, we learned about a methodology for measuring the actual effect of adherence inhibition. The actual organisms used in the iGEM education were provided by Cecil Research.

In the meeting with sessile Research, we were able to learn about the problems with the project's claims and reconfirm the project's strengths. The meeting with Cecil Research also gave us a broader perspective on where to implement our project in society, as we had been focusing only on ships. We were also able to learn from them how to handle sessile organisms when conducting actual research. We would like to express our gratitude again to Cecil Research for their cooperation in this project.

Our research results have a very important impact mainly in the shipping and fishing industries that use vessels. Therefore, it is very important to interview those who will be impacted by our research. We went to the Hirakata fishing port to talk to people involved in the local fishing industry to ask about current problems, the importance of our research findings, and their requests.

As a result, we found that our research had a very significant impact. Current ship-bottom paints are only effective for a very short period, and many of the paints actually used contain copper oxide, which is considered to be harmful to the environment. In addition, we were told that not only ship bottoms, but also the organisms that adhere to the ship's landing sites in harbors can cause holes in the hulls of vessels. We were able to reaffirm that environmentally friendly substances that prevent the attachment of sessile organisms are extremely useful.

We were also able to advance more concrete discussions on the practical application of these substances. For example, we discussed how and where to apply the paint. There are various types of ship-bottom paints, including those for ship bottoms and propellers. The paint needs to be tailored to the characteristics of the vessel. In addition, we felt the possibility of developing new types of paints, such as paints to be applied at harbor berths.

About barnacles, which are the subject of our research on adhesion inhibition, we had not ascertained how the substances that induce adhesion and the inhibiting substances act, and the effects of barnacles as an invasive species due to their transport. In addition, it was unclear how to increase the feasibility of processing into paint, we have in mind as a goal. Therefore, we spoke with Kado Ryusuke , a researcher at the Marine Ecology Research Institute, and Yasuyuki Nogata, a researcher at the Central Research Institute of Electric Power Industry, who are conducting research related to marine sessile organisms and inhibition mechanisms. They told us that the mechanism of the adhesion-inducing substances is not well understood and is still in the realm of hypothesis. As for the mechanism of adhesion-inhibiting substances, which are artificial act on nerves and their toxicity effect. On the other hand, about the natural inhibiting substances, there are many unanswered questions partly because research on barnacles themselves has not progressed, he said. With so little information available on inhibition, there is no choice but to identify inhibitory substances through screening surveys. In addition, there have not been many reports on the impact of the species on the invasive species, and the fact that there are few researchers may be a reason for the paucity of reports. Also, about processing into paint, he suggested that it would be possible to see if the inhibitors can maintain their activity by mixing with paints and to see if they have an affinity for the paints in the short period of time available for research.

Since there were many unknowns regarding the mechanism of action of the inhibitor, it was found necessary to conduct more practical research rather than just considering the problems when we process the substances into paint.

While economic losses and environmental problems caused by barnacles adhering to ship bottoms and interfering with running are well known, there are few reports on the effects of barnacles as an invasive species, which can be assumed from the fact that barnacles are carried on the bottoms of ships. This information was also obtained from interviews with a researcher at Tokyo University of Marine Science and Technology. Therefore, to confirm whether such a problem exists by conducting a more detailed survey of experts, we interviewed Mr. Michio Ohtani, who has been researching the impact of non-native species in the ocean, particularly barnacles among the marine sessile organisms.

Of the non-native species brought in by ships, 70% are introduced by adhesion to the hulls of ships, and many of the non-native species of barnacles are also adhered to the hulls of ships. In addition, we learned that there were cases overseas where oysters have died in areas where non-native barnacles have been introduced, and the ecosystem has been affected. However, these are only a few cases, and most of the problems caused by barnacles are the economic losses and environmental problems. The economic loss is not limited to fuel and cleaning costs, but also includes the costs associated with shutting down operations.

Other organisms known to adhere to ships include the blue mussel, but the blue mussel is less adherent, and barnacles, which can adhere to flat surfaces, are thought to have a greater impact on ships than blue mussels. It is also assumed that the combination of antifouling paint and water currents is effective in inhibiting barnacle adhesion, and it will be a future challenge to improve the ability of antifouling paint to inhibit adhesion even in a stationary state. The fact that barnacles have a significant impact on vessels is a problem that needs to be solved, and this study was again highly beneficial. We also got a hint that the key to prospects is the effectiveness in the absence of water currents.

For the paint we are ultimately aiming for, we lacked detailed information on its mechanism of action and the amount of inhibition we should aim for. We also did not know whether corrosion would be caused by barnacle adhesion. Therefore, we interviewed Dr. Shinichi Motoda of Tokyo University of Marine Science and Technology, who studies antifouling paints and metal corrosion, and asked the mechanism of action, detailed role, and amount of inhibition of adhesion inhibitors.

He told us that no corrosion or erosion occurs due to barnacle adhesion itself. Antifouling paints are developed according to the situation in which they are used, and most of those currently in use are designed for use in seawater with water currents. The adhesion inhibitors are designed to keep the ship completely free of marine sessile organisms, and the ships are re-coated about approximately once every two years. In addition, the substance we focused on is a peptide, and since barnacle larvae use protein-based substances to adhere to something, the substance is expected to be effective in inhibiting larval adhesion.

The effectiveness of the paint is basically determined by such factors as whether the adhesion is controlled to the extent that it does not interfere with the running of the ship. That is based on hydrodynamic analysis of data obtained through experiments in which the material is exposed to seawater and water currents.

Furthermore, we got information that silicon and polymers are now beginning to be utilized as inhibitors.

Since we were informed that in most cases, the barnacles' adverse effects in relation to the ship were related to its running ability, we were able to determine that it would be appropriate to use the resistance of the water flow and similar factors as indicators of evaluation when ascertaining their effectiveness.

In addressing the problem of biofouling by biosynthesizing Cyclo-L-Trp-L-Ala (cWA), it was unclear how to increase the amount of the substance synthesized. Therefore, we had discussions with Professor Naoki Takaya of the University of Tsukuba's Institute of Life and Environmental Sciences. Through that, we found out that increasing the synthesis of cWA is highly feasible. Specifically, cWA can be synthesized based on two types of amino acids, Ala and Trp, as substrates, and if the expression of these amino acids can be increased in E. coli cells, we can get more cWA biosynthesis and synthesis in amount. It also became clear that our project is very significant in terms of synthetic biology. Substances currently used as adhesion inhibitors are reported to contain toxins in their degradation products, so they could be regulated in the future. The biosynthesis by Ala and Trp, which are common substances in living organisms, will make it possible to synthesize substances that are effective in inhibiting adhesion at low concentrations, which neither produce toxins nor affect the ecosystem and the sessile organisms themselves. Industrial synthesis of cWA is often said to be costly. However, the biosynthesis of cWA using the materials in living organisms is conducted in a relatively simple route, so it can be inferred that the cost of synthesis will be reduced. Previously, we had also considered the biosynthesis of Barettin, an analog of cWA, but through the discussion, it was suggested that Barettin has multiple synthesis phases and that biosynthesis is more difficult, so we decided that the ultimate goal was the biosynthesis of cWA. Furthermore, this research was highly evaluated to work in Japan, where the precision fermentation industry is thriving. Mr.Takaya also told us that the biosynthesis of cWA has potential applications in other fields, such as medicine, as a biologically active substance. Based on this discussion, we decided to increase the synthesis of Ala and Trp, and as a first step, we started genetic manipulation to increase the amount of Ala in the cells. In addition, Mr. Takaya's advice on the possibility of application was very helpful in our search for further Human practice sites for our project.

On May 29, 2023, we discussed with Professor Hideo Kigoshi of the Department of Chemistry, Institute of Pure and Applied Sciences, University of Tsukuba, two points from a chemical perspective: the experimental method for implementing antifouling paint which contains cyclo-L-Trp-L-Ala ("cWA") and the extraction method of cWA after cultivation. 1. Implementation experiments of antifouling paints containing cWA He taught us the importance of reagents and the feasibility of the implementation experiment. We decided not to include it in this protocol because it would be difficult in our research span in terms of the cost of reagents and the length of the implementation experiment. 2. Extraction cWA is a polar molecule. Therefore, it dissolves in alcohol but not in hexane. When we asked about extraction methods, we received the suggestion that hexane can be used since it is not necessary to be completely soluble when implemented as a paint. Since there are limited extraction methods in the laboratory, we decided not to use hexane extraction after further discussion, but to use gel extraction by electrophoresis. 3. Others Dr. Kigoshi told us about a website that will help us plan our experiments. The site is called "SciFinder," which is a search tool for chemical reactions and synthesis. We were also able to use it free of charge through our university account and made good use of it in subsequent meetings.

The Japanese Society for Bioscience, Biotechnology, and Agrochemistry (JSBCA) "Challenge Your Dream" Award

We submitted our research plan for this project to the "Challenge Your Dreams Research Project Award" of the Japanese Society for Bioscience, Biotechnology, and Agrochemistry (JSACA). As a result, our project plan passed the document screening. After that, we had three opportunities to discuss our research plan with various researchers belonging to the Japanese Society for Bioscience, Biotechnology, and Agrochemistry (JSACA) before presenting our research plan at the JSACA meeting.

I received various advice from the JSAC. The following is a summary.

Favorable comments

• The social impact of a successful project will be very large.
• The theme can contribute to the SDGs.
• The theme and its conception are novel.

On the other hand, we also received many comments on the problems of the project and the problems with the story in terms of appealing the research plan.

• There are cost problems in effective concentration and production of microorganisms that inhibit adhesion.
• There are many previous researchers in this field, and there are some concerns about patent issues and feasibility.
• It is necessary to show the superiority over chemical synthesis.
• There are problems in social implementation.

We held a total of three meetings to discuss these issues. The cost issue was not the main topic of discussion due to the difficulty of quantifying the cost of microbial synthesis. As for the advantages over chemical synthesis, the high cost of cWA synthesis became clear during the discussion. Furthermore, Dr. Takatani advised that an approach from synthetic biology could increase the amount of synthesis, as in the previous meeting with Dr. Takatani. Regarding the issue of social implementation, he advised that we need to better understand the physical properties of cWA.

We synthesized our discussions up to this point and gave a 5-minute research pitch presentation at the industry-Academia-Government Academic Exchange Forum of the Japanese Society for Bioscience, Biotechnology, and Agrochemistry (JSACA). The judges suggested that in social implementation, a jelly-like substance containing cWA could be created and floated in harbors. In addition, the use of peptides as an adhesion inhibitor and their environmental benefits were highly evaluated. In July 2023, we had an additional meeting to discuss future research directions. This forum was attended by over 100 people and was a good opportunity to publicize our approach to synthetic biology. The presentations are now available on the website of the Society of Biotechnology and Agrochemistry of Japan.

The discussion at the JSAC, which was triggered by the Dream Challenge, was very useful in considering the direction and appeal points of the research, as well as its novelty and feasibility. It also served as an opportunity to raise awareness of the social implementation of this research and led to the subsequent simulation of the evaluation of the physical properties of cWA.