Abstract

We shared the details of the Human Practice activities and their outcomes with our entire team from the start of the project to the present day. Throughout the project, we consistently reflected on what we had learned or gained from them. These activities and the project are fully integrated, maintaining a closely interrelated relationship where each provides feedback. The Human Practice activities were divided into four sections, each with a distinct theme, and these sections included several vital activities that determined the project's direction (Fig. 13.1.1):

Section 1. Encountering the challenges of plastic recycling
Section 2. Development of the BIND-PETase Module (BIP Module)
Section 3. Proposal for the ElectroCount Biosafety Module (ECB Module)
Section 4. Re-examination of where PET TWINS can be implemented in society

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Fig. 13.1.1. Close relationship between HP activities and projects

Additionally, we conducted a particularly distinctive activity that set a precedent for other iGEM teams: a collaborative live stream with a VTuber (Virtual YouTuber) (to Contribution). As part of our Human Practice activities, we looked for ways to hold discussions with the public, which led us to this unique collaboration. The interactive nature of the VTuber live stream was an ideal real-time platform to engage with the audience.　This collaborative livestream was a pioneering endeavor for any iGEM team; it introduced an innovative way to interact with society in the modern era.

Furthermore, we documented all of our efforts in the following sections. They provide insight into how our Waseda-Tokyo team approached the project and the motivations behind our decisions. This documentation also details the background and rationale behind our work, including prior research. Notably, the Experiment Guide we prepared for a laboratory class is one document that future iGEM teams may find helpful. By using this Guide, others can build upon the two-way dialogue with the public, which we initiated, on the issue of microplastics (to Contribution).

Finally, the interviews we conducted with diverse stakeholders shaped our work (Fig. 13.1.2). This holistic approach helped prevent the project from moving forward with a biased perspective and allowed it to grow into something that benefits all members of our society.

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Fig. 13.1.2. Diverse stakeholders involved in Waseda-Tokyo projects

This documentation shows how our Human Practice activities have allowed us to integrate multiple perspectives from all levels of our local community. Hence, we can confidently state that we have made a significant contribution to creating a project that is both responsible and good for the world.

Interactions

Section 1. Encountering the challenges of plastic recycling

What we did in Section 1.

We became aware of the seriousness of the microplastic problem while preparing for the UniLab laboratory class. Following subsequent research, we developed an interest in the recycling of waste plastics. We thought that collecting and processing waste plastics to minimize microplastic outflow could prevent microplastic pollution from worsening. Moreover, we believed establishing effective collection methods could significantly reduce the amount of microplastics in the environment. To achieve this, we concluded that expanding the use of ecological material recycling methods would be crucial for adequately handling waste plastics.

For this purpose, we recognized the need to specifically target PET. The reason is that this particular type of plastic significantly reduces the efficiency of material recycling for composite-material plastics—products made from multiple types of plastic—and lowers the quality of the resulting pellets. Therefore, we focused on PETase as a potential solution to address this challenge and decided to investigate this enzyme.

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Fig. 13.2.1. Section 1. Encountering the challenges of plastic recycling

Fig. 13.2.2. Preparation day and day of UniLab

UniLab

Becoming aware of the severity of the microplastics problem

UniLab_Reflection

While UniLab itself ended as a success, rather than feeling accomplished for teaching the students, we realized how much we underestimated the severity of the microplastic problem. While preparing for the event, we encountered a level of microplastic pollution that was far beyond what we had expected.

Initially, we assumed that we would be filling a bucket with sand containing a few pieces of microplastics for the students to search for. However, when we visited a nearby beach and simply looked at the ground, we were appalled by the overwhelming number of microplastics that were scattered all over the sand. The variety in the colors and shapes of the microplastics spread across the beach made it seem impossible to retrieve them all.

Reaffirming the gravity of the issue, we decided to investigate whether any initiatives were being taken to collect microplastics from beaches and oceans.

Background & Content

We organized and taught a laboratory class at an event called UniLab, which stands for University Laboratory. This science event is organized annually by the School of Science and Engineering at Waseda University for elementary and middle school students.

The Waseda-Tokyo laboratory class focused on microplastic pollution in the oceans as a pressing environmental issue. We engaged our students in discussions about how we can address this problem. We chose microplastic pollution as our topic because it is a significant concern for Japan, which is an island nation. Furthermore, several team members, including our team leader (a dedicated scuba diver), have developed an interest in marine environmental issues.

In the class, the students searched for microplastics in samples of sand that we collected from Tokyo Bay and observed them under a microscope. We then gave a lecture on the characteristics of microplastics and the complications they cause. This was followed by another lecture on synthetic biology. Afterward, students received sticky notes of different colors. They were asked to write down and share with their peers their preexisting knowledge on microplastics, what they learned during the class, and what they wanted to explore further based on what they had learned.

Fig. 13.2.3. Interview with SUSTAINABLE JAPAN

SUSTAINABLE JAPAN

Current Microplastics Countermeasures: Realizing that complete removal of microplastics is impossible

SUSTAINABLE JAPAN_Reflection

This interview revealed that collecting all microplastics released into the environment is currently impossible. This limitation arises from the fineness of the collection equipment's mesh and the restricted options for installation sites.

We also learned about a type of plastic recycling method called material recycling, which piqued our interest. Material recycling is the process of melting waste plastics into pellets, and then using those pellets to create new plastic products. SUSTAINABLE JAPAN mentioned their future plan to utilize this process to recycle the plastics collected by their devices. As a result, we concluded that engaging in material recycling would contribute to a more circular economy.

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Fig. 13.2.4. Material Recycling

Background & Content

We interviewed SUSTAINABLE JAPAN. They domestically sell Seabin, a device developed by an Australian startup for collecting marine plastic debris. They have also created a device called SEETHLIVER that collects plastic waste produced by coated fertilizer membranes floating in drainage channels. During the interview, we explored the benefits and challenges of using these equipment for plastic waste collection. We also discussed the company’s future plans.

However, through the interview, we learned about the limited nature of installation locations for Seabin and SEETHLIVER. While these devices can collect microplastics that are larger than a specific size, they struggle to capture smaller microplastics. This is because if the mesh is made small enough to accommodate these smaller microplastics, it would obstruct an essential procedure of water drainage.

In addition, the plastics collected are treated as waste and not reused. Still, SUSTAINABLE JAPAN mentioned future plans to manufacture and sell plastic products from the collected plastics—a process known as material recycling.

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Fig. 13.2.5. Seabin and SEETHLIVER ^[1]

Fig. 13.2.6. Visit to the Ministry of the Environment

Ministry of the Environment

Becoming aware of the importance of preventing microplastic outflow

Ministry of the Environment_Reflection

From our interview, we realized that it would be more effective to prevent the outflow of microplastics into the environment in the first place rather than to improve methods of collecting microplastics. Due to the limitations of collecting microplastics and the need for proper quantification methods, it is impossible to remove them entirely once released into the environment. Therefore, we thought that by reducing the amount of microplastics generated from waste plastics, rather than relying solely on collection, we could reduce the net change in the outflow of microplastics against those collected as a whole to a negative amount.

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Fig. 13.2.7. Net change between outflow MPs and collected MPs

From here, the team turned its attention to the recycling of waste plastics to prevent microplastic outflow. There are multiple sources of microplastic outflow, but the best-known cause is the weathering of waste plastics. To prevent this, we need to reduce the rate of illegal littering and increase the proportion of waste plastics that are appropriately handled. Therefore, we turned our attention to recycling as an appropriate way of disposing of waste plastic, with the idea of reducing the amount of microplastic outflow in the future by extending the scope of recycling. In particular, we decided to contact companies that deal with material recycling projects to learn more about material recycling, following our sparked interest after interviewing SUSTAINABLE JAPAN.

Background & Content

To understand the international perspective of the microplastics issue, we visited the Ministry of the Environment’s Office of Policies against Marine Plastics Pollution. The Ministry of the Environment is the driving force behind the Japanese government's overall environmental policies. They conduct national-level research and implement measures to address a variety of environmental issues.

What we found particularly interesting in our interview with the Ministry of the Environment is that there is no defined risk assessment and quantification method for microplastics, which vary in size and type. In other words, easy recognition of microplastics is complex in the event of outflow.

Fig. 13.2.8. The interview with esa

esa

Finding the challenge: benefits and problems of material recycling of waste plastics

esa_Reflection

Through our interviews with esa, we became convinced that material recycling is the appropriate way to dispose of waste plastics. As summarized below, thermal recycling emits large amounts of CO2. Chemical recycling also has problems, such as the need for chemicals and CO2 emissions from the recycled products. Therefore, material recycling is an eco-friendly method compared to these other forms of recycling.

Thermal Recycling

A recycling method that involves incinerating waste plastics and other materials. The heat is used to recover electricity.
It is commonly known as energy recovery and is often not accounted for as recycling.
In Japan, thermal recycling processes approximately 5.1 million tons of waste annually. ^[2]
This process accounts for 72% of all plastics recycling in Japan. ^[2]
In Japan, thermal recycling emits approximately 14 million tons of CO2 (CO2 emissions from the combustion of waste plastic equates to 2.76 tCO2/t ^[3]).

Chemical Recycling

A recycling method in which waste plastics are converted back into monomers or chemical raw materials, or used as gas, fuel oil, or coke.
It accounts for 3% of all plastic recycling carried out in Japan. ^[2]
These processes often require the addition of separate chemicals.
The use of the resulting fuel oil and coke further emits CO2.

Material Recycling

A recycling method in which waste plastic is crushed and heated to make pellets. The resulting pellets produce new plastic products.
It accounts for 25% of plastic recycling in Japan. ^[2]
Processing composite-material plastics is difficult due to differences in the melting points of the respective materials.
This process reduces the energy efficiency of pelletizing and the quality of the resulting pellets. This is especially true if the plastic contains PET, which has a high melting point.

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Fig. 13.2.9. Percentage of plastic recycling methods practiced in Japan^[2]

We then thought about selectively removing PET in composite-material plastics to reduce material recycling costs and to increase its scope of applicability. esa says PET, which has an exceptionally high melting point among commonly used plastics, negatively impacts material recycling composite-material plastics. Therefore, the energy efficiency of pelletizing and the quality of the resulting pellets could be improved if only PET could be selectively removed from them.

We focused on PETase as a means of explicitly degrading PET in composite-material plastics. In order to further investigate the use of PETase, we decided to conduct interviews with companies and organizations that are considering the industrial use of PETase.

Background & Content

Next, we interviewed esa. Three types of plastic recycling methods are recognized in Japan: thermal recycling, chemical recycling, and material recycling. esa specializes in material recycling.

During the interview, we asked about the advantages and challenges of material recycling. Material recycling is a method in which waste plastics are melted down and pelletized to make new plastic products.

Generally, waste plastics are divided into single-material plastics made from one type of plastic and composite-material plastics made from multiple types of plastics. According to esa's estimation, composite-material plastics account for approximately 25% of the total waste plastics. Processing composite-material plastics by material recycling was difficult in the past. However, recent advancements in technology have opened doors to pelletizing these materials. Still, there is no doubt that the material recycling of these plastics is complex. In particular, processing composite-material plastics containing PET is expensive and lowers the resulting pellets' quality.

Section 2. Development of the BIND-PETase Module

What we did in Section 2.

To explore the use of PETase for selectively removing PET from composite-material plastics, we investigated the current status of PETase's social implementation. After identifying issues with PETase, we focused on the BIND-system as a potential solution. Our goal was to obtain BIND-PETase(WT) mutants with enhanced functionalities. We selected superior mutants by conducting both simulations and laboratory experiments. Then, we performed degradation experiments on PET-related pellets.

Finally, having developed the BIP Module, we gathered feedback from industry professionals regarding the remaining challenges behind BIND-PETase. We decided on our next course of action based on their insights.

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Fig. 13.2.10. Section 2. Development of the BIND-PETase Module

Fig. 13.2.11. Interview with OpenPETase

OpenPETase

Recognizing the barriers to PETase's social implementation

OpenPETase_Reflection

From our interview with OpenPETase, we realized that the direct utilization of PETase may not always be the best solution. Although PETase is an enzyme gaining attention in many research laboratories, it has seen limited practical use. This is due to the following reasons:

Difficulties in achieving high yield and quality during purification
Challenges in maintaining enzyme activity over time
The enzyme can only be used once

To tackle these limitations, we focused on BIND-PETase ^[4]. Unlike free PETase, BIND-PETase displays PETase on the surface of cells, so it eliminates the need for purification. Additionally, it offers several other advantages (to Design BIP Module).

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Fig. 13.2.12. BIND-PETase Module graphical abstract

Background & Content

To understand the current state of PETase’s social implementation, we interviewed OpenPETase, a nonprofit organization focused on leveraging PET degradation by PETase for social contribution.

In the interview, we first asked for more details about OpenPETase’s project. Their work mainly focuses on developing a PET-degrading device using PETase. However, they are also exploring alternative solutions, such as plant-based polymers that can decompose in soil, and wax worms, which are bacteria that can degrade plastics. One of the main reasons they are not solely focused on PETase is its challenges in being efficiently purified under controlled laboratory conditions. This fault makes mass production of PETase difficult.

Fig. 13.2.13. Prof. Onodera

Prof. Onodera

To obtain a more efficient BIND-PETase mutant

Prof. Onodera_Reflection

Through discussions with Professor Onodera, we determined that conducting directed evolution within the scope of iGEM was not feasible due to time and cost constraints. As a result, we decided to shift our focus to designing mutants using machine learning while continuing to consider rational design as an alternative approach. Additionally, based on Professor Onodera's feedback, we recognized the limitations in the reliability of designing mutants with machine learning.

To address this, we performed in silico screening—computer-based screening—to computationally replicate the selection pressures of directed evolution. Regarding the fusion protein between CsgA and PETase that we are designing, we identified three key evaluation criteria:

CsgA has to be able to form polymers
PETase must have the activity to degrade PET
CsgA and PETase must retain stability upon fusion

Background & Content

We aimed to design BIND-PETase (WT) mutants with enhanced activity, thereby expanding BIND-PETase’s potential for industrial application. While many studies have focused on improving the functionality of PETase, they primarily used the wild-type PETase (WT) from *Ideonella sakaiensis*. To apply the findings of such studies on PETase to BIND-PETase, we began an evolutionary engineering project to optimize BIND-PETase.

We sought advice from Professor Wataru Onodera. He is an expert in evolutionary engineering at Waseda University's School of Advanced Science and Engineering. He helped us deepen our understanding of the characteristics of two approaches: directed evolution and rational design.

Fig. 13.2.14. Prof. Yura

Prof. Yura

Proposal for the approach to in silico screening

Prof. Yura_Reflection

Through consulting with Professor Yura, it became clear that executing MD simulations with our computational resources would be challenging. Additionally, due to the low accuracy of the csgA-csgA docking, we decided to shift our focus to the small molecule docking of PET-PETase.

Background & Content

In response to the evaluation criteria identified above, we proposed the following experiments: protein docking of csgA-csgA, small molecule docking of PET-PETase, and molecular dynamics (MD) simulations. However, while progressing with these simulations, we encountered technical challenges. We consulted Professor Takashi Yura, who is knowledgeable in molecular dynamics and docking.

The specific consultation topics are as follows:

Setting up MD simulations
Reasons why the csgA-csgA protein docking is not progressing well

Fig. 13.2.15. Shizuku Ph.D.

Shizuku Ph.D.

Proposal for the Molecular Docking Flow

Shizuku Ph.D._Reflection

Shizuku Ph.D. suggested that it is difficult for beginners to assess docking accuracy visually. We were therefore recommended to use an electron density map for evaluation. Additionally, we learned that since PET is a polymer, the docking accuracy would vary depending on its size. Therefore, we decided to perform docking of 2PET and 4PET, and evaluate it using an electron density map. Furthermore, since evaluating all the candidates generated by machine learning through docking is challenging, we proposed filtering them in advance based on scores.

Based on these interviews, we constructed an In Silico Evolution Pipeline. This pipeline consists of three steps: "mutant design using machine learning," "in silico screening," and "human validation." Using this pipeline, we designed five mutants that enhance the function of BIND-PETase (WT) (to Model Simulation, Engineering Success). We also worked on rational design, creating nine additional mutants. Subsequently, we validated the functionality of the mutants through experiments and selected the most promising Composite Part as "RBS+BIND-bearPETase" (BBa_K5436124). For more information, visit the Registry page.

Background & Content

We succeeded in the docking of PET-PETase. Still, we faced the challenge of evaluating the accuracy of the docking. Therefore, we sought advice from Shizuku Ph.D., an expert in the pharmaceutical field.

Fig. 13.2.16. Meeting with people from Fermelanta, Inc.

Fermelanta, Inc.

Reconfirming that the purification-free nature of BIND-PETase is a major advantage

Fermelanta, Inc._Reflection

During our discussion on the social applicability of BIND-PETase, we gained insights on the following points:

The purification-free nature of BIND-PETase is highly cost-saving
The scale of scale-up should be envisaged

From this feedback, we reaffirmed that the BIND-system has high potential as a critical solution for the industrialization of PETase, which further boosted our confidence.

Background & Content

We had the opportunity to interview Fermelanta, Inc., a synthetic biology start-up based in Ishikawa Prefecture, Japan. They specialize in the microbial production of rare components derived from plants. They possess expertise in the production of valuable substances by microorganisms and the improvement of their efficiency.

We consulted with Fermelanta, Inc. to get an objective opinion on whether using the BIND-system for the social implementation of PETase would be worthwhile.

Fig. 13.2.17. PET-related pellets provided by esa

esa (2nd Interaction)

Degradation of PET-related pellets provided by esa

Background & Content

We were offered PET-related pellets as experimental samples from esa. After our interview about material recycling, we informed our intention to work on the specific removal of PET, and they promised to provide PET-related pellets if necessary. After selecting high-performing BIND-PETase mutants, we conducted PET degradation experiments on the actual pellets. The BIND-PETase used in the experiments can be found here (to Engineering Success). In addition, the details can be checked here(to Results).

Section 3. Proposal for the ElectroCount Biosafety Module

What we did in Section 3.

We investigated sterilization methods for genetically modified organisms to overcome the issues related to BIND-PETase, which uses E. coli as a whole-cell catalyst with PET-degrading activity. Based on our investigations, we were advised that it is crucial to combine multiple sterilization methods. Hence, we considered combining two sterilization approaches: the expression of the MazF protein and applying over-voltage.

We implemented electric control to enable MazF expression at any desired time. We contacted experts in electrochemistry to discuss the experimental system. Moreover, after we could not obtain the expected results in some experiments, we scheduled an interview with the author of the original reference paper we consulted. Then, to examine the exact experimental conditions explained during the interview, we first performed simulations.

We constructed a device called PET TWINS from these tasks, consisting of a BIP Module and an ECB Module. Then, we finally revisited the question of finding the most applicable location for this device's social implementation.

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Fig. 13.2.18. Section 3. Proposal for ElectroCount Biosafety Module

Fig. 13.2.19. The interview with YAEGAKI Biotechnology

YAEGAKI Biotechnology

Strongly recognizing the need for sterilization as an issue with BIND-PETase

YAEGAKI Biotechnology_Reflection

Through the interview with YAEGAKI Biotechnology, we decided to consider the use of the suicide gene MazF. While industries outside the food sector that utilize genetically modified organisms can operate actively in Japan, sterilization methods still require careful and thorough discussion. Since heating the outer perimeter of the plant can be costly, we thought that achieving further efficiency and cost reduction would greatly reduce the burden on companies. With our attention caught by MazF, we shared the necessity with our team to investigate the sterilizing potential of this suicide gene.

Background & Content

After developing the BIP Module, we interviewed YAEGAKI Biotechnology to gather on-site feedback regarding the challenges of incorporating BIND-PETase in an industrial setting. Since BIND-PETase utilizes *E. coli* as a whole-cell catalyst with PET-degrading activity, there are concerns about the leakage of genetically modified organisms. YAEGAKI Biotechnology is a company involved in fermentation-related businesses that utilize microorganisms for industrial means.

According to YAEGAKI Biotechnology, as a general public perception, the industrial use of genetically modified organisms is actively permissible in Japan, provided it is not for food. However, they advised that management of the microorganisms must be strictly enforced from a biosafety perspective, especially regarding sterilization methods. They also noted that while heating the tank’s outer perimeter is a major sterilization method, it is also costly, so it would be even better if an alternative sterilization method could be implemented instead.

Fig. 13.2.20. Interview with Prof. Tsuneda

Prof. Tsuneda

Recognizing the inadequacy of MazF's sterilizing capacity and the importance of layering multiple sterilization methods

Prof. Tsuneda_Reflection

Following our discussion with Professor Tsuneda, we were able to consider the utility in the social implementation of the MazF gene, which is as follows:

MazF aims to inhibit the proliferation of E. coli, but it cannot eliminate them
MazF alone does not establish complete biosafety
A combination of multiple sterilization methods is necessary to ensure biosafety

Therefore, to contribute to biosafety, we decided to not only express MazF but also incorporate other means of sterilization. We began investigating unique and low-cost sterilization methods beyond heat sterilization and MazF expression.

Background & Content

During our interview with YAEGAKI Biotechnology, we learned that low-cost sterilization procedures are essential for the industrial use of microorganisms. Furthermore, in the case of BIND-PETase technology, where genetically modified organisms are directly socially implemented, it is necessary to have a detailed understanding of the reliability of sterilization techniques.

As a result, we investigated using the MazF gene, a typical kill switch gene, for bacterial sterilization. To deepen our understanding of MazF's sterilizing ability, we spoke with Professor Satoshi Tokuda from Waseda University's School of Advanced Science and Engineering. His lab has been testing various MazF gene mutants and has accumulated extensive knowledge of their sterilizing properties.

We were able to ask the following questions regarding the MazF gene during the interview:

What is the time scale until cell death is induced since MazF is expressed?
Quantitatively, what is the degree of its sterilization capability?
If the sterilization by MazF is incomplete, what other measures should be taken?

Fig. 13.2.21.

High electric field pulse sterilization ^[5]

Investigation

Investigation of over-voltage sterilization

Result

While investigating sterilization methods, we discovered the existence of over-voltage sterilization. Over-voltage sterilization refers to sterilization achieved through high-voltage pulses. It has a straightforward mechanism: first, a high electric field pulse is applied to the cells. This application induces the movement of ions in and out of the cytoplasm, generating a potential difference across the cell membrane. The Maxwell stress generated by this potential difference compresses the cell membrane, leading to its breakdown. ^[5] This discovery prompted us to combine MazF with over-voltage sterilization to enhance biosafety.

We then decided to investigate the electric regulation of gene expression in E. coli. This electric regulation is necessary because we need to express BIND-PETase and MazF at specific times. In other words, we must sufficiently degrade PET with the expressed BIND-PETase before sterilization. To address this problem with over-voltage sterilization, we initiated our research on the electric control of gene expression in E. coli.

Fig. 13.2.22. Electrical control concept for bioremediation in the early stages

Investigation

Investigation of electric regulation of gene expression

Result

We considered electric regulation for controlling PETase expression to expand its industrial applicability. Conventional microbial control methods, such as chemical stimulation and optogenetic control, have been tested by many iGEM teams in their quest for industrialization. However, chemical and optogenetic controls have the following disadvantages.

Disadvantages of chemical stimulation:

It is challenging to automate control.
Once the chemicals are added, the stimulation is continued. This continuation makes it difficult to control multiple reactions.

Disadvantages of optogenetic stimulation:

Restricted use in enclosed or dark environments, limiting a number of potential implementation sites.
In tanks with significant volume or turbidity, the stimulation may not extend to the entire culture medium, making complete control difficult.

In contrast, electrical control offers the following advantages.

Advantages of electrogenic stimulation:

More straightforward automation of control.
Stimulation is not continuous, allowing multiple stimuli to be applied to the gene circuit using a single type of stimulus. This enables more complex reactions.

To take advantage of these benefits, we initially considered placing the BIND-PETase gene downstream of the electrically inducible promoter characterized by the iGEM 2022 Paris_Bettencourt team^[6] and Joshua Laurence^[7] (Fig. 13.2.22).

Moreover, we leveraged the benefit of the electrical control's "ability to drive complex responses with only one type of stimulus, eliminating the need for continuous stimulation." We aimed to use this advantage to ultimately construct an ECB Module (ElectroCount Biosafety Module). For a detailed description of the ECB Module and the promoter mechanism, please see the Design and Description tabs.

Next, we decided to contact researchers specializing in electrochemistry to develop a suitable experimental system for conducting lab-scale electrical response experiments.

Fig. 13.2.23. In conversation with Prof. Lawrence

Prof. Lawrence

Understanding the experimental system and starting the simulation

Prof. Lawrence_Reflection

We received the following advice throughout our interviews.

The wild-type pSoxS is a leaky promoter, so if we use it to control the expression of toxic proteins like MazF, we should consider using a mutant with less leakage.
The gene circuit can be made into a NOT circuit to prevent the initiation of gene expression by spontaneous oxidation.

We then investigated the experimental conditions through simulations (to Model Simulation). This was because the interview provided us with a better understanding of the properties of pSoxS and the rigorous experimental conditions we need to consider for conducting electrical response experiments.

Background & Content

To build the ECB Module, it is essential to fully understand the nature of its central electrically inducible promoter. Therefore, we spoke with Joshua Lawrence, who authored a paper demonstrating the electrochemical induction of pSoxS through the application of voltage.

During the interview, we were able to ask him about the following:

What characteristics does the pSoxS promoter have?
What should be kept in mind when conducting experiments with electrically inducible promoters?

Fig. 13.2.24. Prof. Nakanishi

Prof. Nakanishi

Examining the experimental system with an electrochemistry expert

Prof. Nakanishi_Reflection

In our discussion with Professor Nakanishi, we received the following advice:

Looking ahead to our future experiments, we should concretize our experimental plan and construct our experimental system.
A power supply is essential regardless of the kind of experimental system we construct.

In response, we further refined our experimental plan and extracted the necessary elements. Based on this, we further discussed with Dr Nakanishi to decide on the specific products to purchase. As the power supply was expensive, we decided to build it ourselves (dry lab hardward). Furthermore, we also received help in measuring the redox potential of pyocyanin, which is necessary for the electrical response (wet lab results 9.7).  Using the experimental system thus constructed, we carried out experiments on the electrical response.

Background & Content

To conduct experiments on electrical responses, we had to construct an experimental system that would adequately conduct electricity in the culture tanks. Therefore, we decided to interview someone with extensive knowledge of electrochemistry and experience in electrochemical experiments. We interviewed Professor Takaya Nakanishi, a senior researcher at Waseda University’s Comprehensive Research Organization.

We asked the following questions in the interview:

Is it possible to apply voltage to the culture medium hourly using standard laboratory equipment?
If not, what equipment do we need to construct the experimental system?

Section 4. Re-examination of where PET TWINS can be implemented in society

What we did in Section 4.

Having built PET TWINS, we wanted to reaffirm that material recycling is the most appropriate application for this device before fully considering its social implementation. For this purpose, we collected opinions from the general public through collaborative distribution and visited municipal management facilities. The results of these actions proved once again that the most appropriate application of PET TWINS is to improve the efficiency of material recycling.

Finally, we discussed the need to consider tank size and overall flow for social implementation and decided to discuss these with the whole team.

alt text

Fig. 13.2.25. Section 4. Re-examination of where PET TWINS can be implemented in society

Fig. 13.2.26. Collaborative livestream with Mr. Takatoh

Mr. Takatoh

New efforts to receive public opinions on PET TWINS' social implementation

Mr. Takatoh_Reflection

After the collaborative livestream, we visited the Water Reclamation Center as suggested. This was partly because it was the first time throughout the project that the idea of installing PET TWINS in a municipally managed facility was proposed. Another reason why we followed through with the suggestion was because we were informed during the live stream about the removal of microplastics from household wastewater at this center.

Background & Content

As we developed PET TWINS, we decided to reevaluate the most suitable options for applying this device to prove that material recycling is the best option. To do so, we planned to gather candid opinions from the public by explaining the concept of PET TWINS and asking where they believe the device could be implemented. This led to a collaborative livestream with a Virtual YouTuber (VTuber) named Rai Takatoh. Mr. Takatoh is a researcher in life sciences at a chemical company. On the side, he regularly shares biology-related content on YouTube.

During the live stream, we first explained our project and provided viewers with an overview of PET TWINS. Afterward, we interacted with the audience in real-time via the comments section. In particular, the question, "Where should a device like PET TWINS be implemented to reduce microplastics?" generated two leading suggestions: businesses and municipalities. One specific example mentioned for municipalities was the Water Reclamation Center.

Fig. 13.2.27. Visit to the Morigasaki Water Reclamation Center

Morigasaki Water Reclamation Center

Final Decision on Implementation Site

Morigasaki Water Reclamation Center_Reflection

After our visit, we concluded that utilizing genetically modified organisms at this Water Reclamation Center would be challenging. This is because, as we received detailed explanations of each stage of the purification process, we determined that it would be difficult to enforce rigorous containment measures for genetically modified organisms at this facility.

With this in mind, we came to the conclusion that material recycling is indeed the most appropriate application for PET TWINS. We were determined to explore the tank size and overall flow necessary for implementation.

Background & Content

We received a tour of the Morigasaki Water Reclamation Center, Japan’s largest wastewater treatment plant, to understand how municipal wastewater is processed and whether there is potential to install PET TWINS there. Located near Haneda Airport, the Morigasaki Water Reclamation Center collects and purifies wastewater from the southern part of central Tokyo before discharging it into the sea.

During our visit, two staff members explained the detailed wastewater treatment process in central Tokyo. The purification process at the center starts with grit chambers and sedimentation tanks to remove large debris and sand from the wastewater. Next, microorganisms break down organic matter, and the treated sludge settles in the secondary sedimentation tanks while the clarified water is extracted. The sludge is then concentrated and digested, gasified to reduce its volume. The methane gas produced is used as fuel within the facility. Finally, the treated water is discharged into Tokyo Bay, reusing some within the center after sand filtration. We also learned that the center is equipped with a facility for chlorine sterilization, demonstrating its environmental consciousness.

Implementation

Our team aims to achieve the on-site breakdown of PET in composite-material plastics—plastic products composed of several types of plastic—using PETase, with PET TWINS. This would allow for the following two recycling options:

Material recycling of composite materials
Material recycling is the most ecological method among various plastic recycling techniques. However, current technology has revealed several issues with recycling composite materials due to different melting points among plastics. In particular, the high melting point of PET significantly reduces the efficiency of material recycling. Therefore, by explicitly breaking down PET in composite-material plastics using PET TWINS, we expect to improve both the efficiency of material recycling and the quality of the resulting pellets.
Recycling of PET → TPA → PET
By recovering TPA obtained from the breakdown of PET by PETase and re-polymerizing it, we can achieve high-purity PET.

Furthermore, we assumed the following flow for the on-site implementation of PET TWINS (Fig. 13.3.1). The first tank is the culture tank, where genetically modified E. coli proliferate under controlled expression conditions. Suppressing the expression of non-essential proteins alleviates the burden on E. coli, allowing for efficient proliferation. The second tank is the reaction tank, where PBS, the composite-material plastics, and the E. coli grown in the culture tank are added. By applying the first electrical stimulation, BIND-PETase is expressed, and PET degradation begins. After the reaction is complete, a second stimulation is applied to kill the E. coli. The composite-material plastics, from which only PET has been removed by the reaction, will be pelletized efficiently in a material recycling plant. In addition, the TPA resulting from the degradation of PET will be stored in a third tank and can be re-polymerized separately to obtain PET once again.

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Fig. 13.3.1. Flow assumptions in PET TWINS implementations

These flow assumptions were instrumental in highlighting the advantages of PET TWINS (specifically in addressing the issues with PETase). In summary, the benefits of PET TWINS include:

Control of expression by electricity
This allows us to only proliferate E. coli without burdening them in the first tank. In the second tank, we can initiate reactions or kill the E. coli at any desired time. This also contributes to biosafety.
No need for purification
Purification is no longer necessary by displaying PETase on the membrane surface of E. coli. This makes it possible to connect the first and second tanks directly. This means that the proliferated E. coli can be used as-is.
Lab automation
In our project, the expression of E. coli can be easily controlled by electricity. The advantage is that this electrical stimulus can be remotely triggered without being physically present. As PET degradation in complex materials is expected to take at least several days, having a system that does not require constant human monitoring throughout the entire process is crucial. The use of electrically inducible promoters will contribute to the development of lab automation, which in the future will allow anyone with a smartphone to control the reaction process at a plant.

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Fig. 13.3.2. Remote control diagram of the plant

Collaboration & Education

Some of our Human Practice activities did not directly impact the main storyline of our project. This section summarizes those initiatives.

Fig. 13.4.1. Discussion with UTokyo

iGEM UTokyo 2024

Becoming aware of the Need for Energy Estimation

iGEM UTokyo 2024_Reflection 

The UTokyo team highlighted several meaningful aspects to us. One was that we were able to confirm the value of our consideration for CO2 in the social implementation of PETase.

We believe that by promoting the on-site use of PETase, we can contribute to material recycling. The leader of the UTokyo team expressed concerns that even if PET is degraded into TPA by PETase, the byproducts, H2O and CO2, would naturally be released—this raises concerns about carbon neutrality. However, with PET TWINS, we aim to minimize CO2 emissions through material recycling. This strengthened our confidence in addressing these concerns.

Additionally, the UTokyo team also performed conformational analysis and molecular dynamics simulations of proteins, making our methods closely aligned. This led to a productive exchange of information. Beyond improving our project, this opportunity allowed us to form meaningful connections with fellow iGEMers, highlighting the significance of collaboration.

Furthermore, the UTokyo team pointed out the need for quantitative justification of the necessity of electrical responses in our project. This inspired us to calculate the energy requirements for electric sterilization and compare the results with those for heat sterilization (to Model Simulation).

Background & Content

After outlining our project and validating its potential through experiments, we held a discussion session with UTokyo (the geographically closest iGEM team). We exchanged details about our projects, providing feedback on points that the general public might find concerning or any risks we should consider from an objective perspective.

Fig. 13.4.2. Interaction at the iGEM Japan Meetup

iGEM Japan Meetup

Collaboration with Other Teams

iGEM Japan Meetup_Reflection

Because we were among people who were also striving for the same stage, during the Q\&A session, we received empathetic voices stemming from shared experiences of similar failures. We also received advice based on the knowledge they gained by overcoming those challenges themselves. Discussing each other’s projects with the other teams confirmed our project's strengths and weaknesses. Based on this feedback, we were able to refine our project further.

Background & Content

At the iGEM Japan Meetup, iGEM teams from across Japan gathered to present their progress on their projects. This event aimed to deepen interactions among Japanese iGEMers and provide feedback on each other’s project contents and directions. As part of our preparation for the Jamboree, we gave our presentation on PET TWINS as closely as possible to the actual presentation conditions. We started our presentation with an introduction to the environmental issue we focused on, followed by a structured report on the research we conducted and the progress we made. Then, we shared the challenges we were facing at the time and outlined our future plans. After the presentation, we responded to insightful questions from members of the other teams and received a variety of feedback.

Fig. 13.4.3. Online events held by AS ONE Corporation

Event with AS ONE Corporation

Gathering Public Opinions on Genetically Modified Organisms

Event with AS ONE Corporation_Reflection

In our event with AS ONE Corporation, we gained valuable insights into public perception of genetically modified organisms. For this event, we did not mention PET TWINS by name; instead, we framed the discussion around a hypothetical device using genetically modified organisms to break down microplastics. This is because we wanted to investigate people's awareness and concerns about genetically modified organisms.

During the event, we polled, "Would you want to install a microplastic degrader that uses genetically modified microorganisms in your home?" To this question, 75% of the participants responded, "No." Through these results and the discussion content, we became keenly aware of the public's concerns about genetically modified microorganisms. This made it clear that, as it stands, implementing devices that use genetically modified microorganisms in homes is a challenging proposition.

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Fig. 13.4.4. Questionnaire result

Background & Content

We had the opportunity to make an appearance in an online event called co-lab BRAINS, organized by AS ONE Corporation. The theme was to introduce Waseda-Tokyo's current research topics, and this presentation was followed by a discussion with the event’s participants about our ongoing work.

The first half of the event was the input part. We first presented an overview of synthetic biology and iGEM. We then shared Waseda-Tokyo's past efforts and finally lectured on the current issues our team is focusing on. In the second half, we held an interactive discussion with the participants. The discussion proceeded in real time, microphones on, primarily focusing on measures against microplastics that can be implemented at home. We exchanged ideas on where microplastics might be residing in our daily lives and what measures we can take to address them starting tomorrow. Through this, we and the participants collectively deepened our understanding of the issue. 

References

SUSTAINABLE JAPAN, https://www.sustainablejapan.org/ ↩︎
Plastic Waste Management Institute (2023)「プラスチック製品の生産・廃棄・再資源化・処理処分の状況マテリアルフロー図」, https://www.pwmi.or.jp/pdf/panf2.pdf ↩︎ ↩︎ ↩︎ ↩︎ ↩︎
Ministry of the Environment (2023)「Santei・hohkoku・kohyo ni okeru santei houho・haisyutu keisuu itiran」, https://ghg-santeikohyo.env.go.jp/files/calc/itiran_2023_rev4.pdf ↩︎
Zhu B. et al. (2022) Enzymatic Degradation of Polyethylene Terephthalate Plastics by Bacterial Curli Display PETase, Environ. Sci. Technol. Lett. 9(7), 650-657, https://pubs.acs.org/doi/10.1021/acs.estlett.2c00332. ↩︎
Yuji M. et al. (2014) Elongation of E. coli after High Electric Field Pulse Application, J. Inst. Electrostat. Jpn.38(2), 108-112, http://www.iesj.org/content/files/pdf/papers/38/38-2-108.pdf ↩︎ ↩︎
Paris_Bettencourt. (2022) Bacterial Networked Electronic Interface. https://2022.igem.wiki/paris-bettencourt/index.html. Retrieved September 30, 2024 ↩︎
Lawrence, J. M. et al. (2022) Synthetic biology and bioelectrochemical tools for electrogenetic system engineering, Science Advances, 8(18), https://doi.org/10.1126/sciadv.abm5091 ↩︎