Through Human practices activities, based on the understanding of the real-world problems faced by the drainage system, we continuously collect the opinions of stakeholders through extensive literature research, field trips, and interviews, and make improvements in the design stage of the project.

SRBioQuencher is a new technology to solve the environmental problems of sewers, needs to be well-designed to adapt to real-world applications. It is always necessary to pay attention to its practicality and feasibility, as far as possible, to design for the specific environment of the sewer, reflecting the relevance of SRBioQuencher.

Based on the Human Practice Cycle and the experience of ourselves, we can roughly divide the process of Integrated Human Practices into 3 phases.

These experiences are in line with the general law of exploration of things and can also be widely applied to the time planning of future iGEM years.

Forming a diverse team

Dec. 2022-Jan. 2023

In late 2022, after member recruiting and interviewing at Sichuan University, SCU-China 2023 was formed, with 21 undergraduate students from various professional backgrounds. In January 2023, we conducted a one-week winter camp for the new and reserve team members. The team members familiarized the process of the iGEM competition and brainstormed the project. These premature ideas were guided and improved by PI teachers and senior students.

Put forward a scientific issue

Feb.-Apr. 2023

We chose the original SRBioQuencher proposal from over a dozen inspirations brainstormed over the winter camp, combining feasibility and innovation among the many ideas put forward by our team members. We initially focused on the problem of H2S and CH4 in drainage pipes. With the entry point of sulfate-reducing bacteria (SRB) and methanogenic bacteria (MA) biofilms, we expected to disrupt and inhibit the symbiotic system of SRB and MA by introducing engineered bacteria.

Solution ver.1

1. Degradation of AHL molecules which introduce biofilm quorum sensing.
2. Inhibit MA by the secretion of triterpene saponins.
3. Forming Bacillus subtilis-E. coli co-culture system to enhance the competitiveness of engineered E. coli in sewer ecosystems.

Ensuring theoretical feasibility

May.-Jun. 2023

May 5^th - Microbiology Specialists

Prof. Jian Zhao and Prof. Nianhui Zhang are experts from the Department of Microbiology, College of Life Sciences, Sichuan University, with whom the team explored the main problems they might encounter in advancing the project.

Problems:

1. Based on our knowledge of the symbiotic system, we lacked a specific target.
2. A better option is to raise the competitiveness of engineered bacteria in the ecosystem. (Bacillus subtilis-E. coli co-culture system?)
3. How to culture MA and SRB and recurrence of the SRB biofilm.

Opinions & helps:

1. SRB is a microaerobic state in SRB-MA symbiotic system while MA is hypoxic. It has been reported that SRB biofilm provides a hypoxic environment for MA, so we can put the target on SRB, and the inhibition of MA as a kind of bonus.
2. E. coli has a certain distribution in urban sewers itself. The co-culture system may not be very meaningful in the rapid water flow in the sewer. We can strengthen the competitiveness of the engineered bacteria by expressing antimicrobial peptides. However, the inclusion of antimicrobial peptides makes the program unfocused, and the amount of expression is also a problem.
3. The anaerobic culture of SRB and MA is difficult, and the pH environment in the pipeline may put pressure on the survival of the engineered bacteria.
4. There is already a well-established biofilm assay, we should consult the Biogas Institute.

Improvements:

1. Preliminary decision to focus on SRB biofilm removal.
2. Planned communication with the Biogas Institute.

Updated solution ver.2

1. Adopt the quorum sensing quenching to inhibit SRB biofilm formation.
2. Inhibit MA by the secretion of triterpene saponins. Through literature review, we discovered the inhibition mechanism of triterpene saponins is unclear, and the application scenario is in the rumen of cattle.
3. Forming Bacillus subtilis-E. coli co-culture system. It's hard to restrict the growth and ensure the biosecurity of Bacillus subtilis.

May 5^th - Molecular Biology Specialists

Prof. Zhibin Liu and Prof. Ying Tong from the School of Life Sciences of Sichuan University have a rich experience in molecular biology research. We hope to hear from their perspectives.

Opinions & helps:

1. Enzymes: Whether the AHL degrading enzyme is still active when tagged to the effluent environment of the drain, and whether the expression level in the engineered bacteria can be improved.
2. Look for the availability of microorganisms that directly consume methane and hydrogen sulfide to ensure our subject would not be replaced.
3. Degrading the existing biofilm in the sewers at the same time to achieve better results.
4. Biosafety problems need to be considered.

Improvements:

1. Furthered inquired about various AHL-degrading enzymes, expected to predict their stability through modeling.
2. Literature review and discussion on whether the program will be replaced.
3. A functional module to disrupt the structure of biofilm was added.
4. Added an AHL-mediated suicide system.

Zhibin Liu

Ying Tong

Updated solution ver.3

1. Added a module to degrade the existing biofilm at the same time: the cocktail therapy.
2. Added a bacteriostatic module by expressing AMP.
3. Designed an AHL-mediated suicide system, which allows the engineered bacteria to commit suicide in low AHL concentration environments.

May 7^th - Bioinformatics Specialists

Yang Cao specializes in bioinformatics and protein modeling. He gave us some comments on the modeling part of the project.

Opinions & helps:

1. AHL degradation enzyme is an exonuclease that requires a high stability, for the enzyme optimization, we need to find similar ones to compare their efficiency.
2. How to make the program more focused with the inclusion of antimicrobial peptides.

Improvements:

1. Point mutation optimization of endostatin (AHL degrading enzyme) based on semi-rational design to enhance the affinity of the enzyme to more AHL subtypes. The validation of the results is firstly done through CB-Dock2 developed by Prof. Yang Cao, and secondly by experiments. In the process of using CB-Dock2, we consulted Prof. Yang Cao for the interpretation of the results and how to better show the results of this part of modeling.
2. Convey the necessity of the bacterial inhibition part by mathematical modeling. We designed a fusion protein of SRB-targeted antimicrobial peptide and broad-spectrum antimicrobial peptide, which improves the competitiveness of the engineered bacterium in the sewage.
3. In the process of suicide system designing, the intensity of each part needs to be carefully considered. The modeling group constructed a differential equation model by expressing each part and fitting the corresponding AHL concentration of the suicide system, which supports us in carrying out the construction of the suicide system in the wet lab.

Updated solution ver.4

1. Based on the bacteriostatic module, fusion proteins were designed to target the microenvironment in which SRBs are located to reduce the impact on other bacteria in the sewer.
2. Simulate the expression of each part in the AHL-mediated suicide system with mathematical modeling to precisely regulate suicide thresholds.
3. All the enzymes used are optimized by semi-rational design, to improve the affinity and the resistance, so that the functional modules can be better adapted to the working environment of sewers.

May 12^th - Engineering Specialists

Prof. Huiqiang Li is an expert in Environmental Engineering at Sichuan University and his main research interests are biological treatment and resource utilization of wastewater. We are looking forward to getting some suggestions based on his expertise, to think better about our involvement in water supply, drainage, and environmental issues.

Problems:

1. How to put engineered bacteria into sewers in a more rational way.
2. How to carry out validation of the effectiveness of biofilm removal by engineered bacteria.
3. How to realize the target therapy of biofilm by engineered bacteria.
4. Whether the removal of sulfate-reducing bacteria will help solve pipe corrosion problems.

Opinions & helps:

1. Sulfate-reducing bacteria (SRB) is a hot topic in the current field of sewer biofilm research. Since MA biofilms need to rely on the anoxic conditions constructed by SRB biofilms, it is appropriate to focus on the removal of SRB biofilms.
2. The environment in the drainage pipe is harsh and complex, the odor is not only from SRB and MA biofilm. We should continue to review the literature to understand the complex flora in the pipeline, considering the interactions between the florae.
3. Embedding and placing the bacterial powder into the sewer is feasible. It is better to select appropriate parameters for modeling based on the basic structure of the water supply and drainage system, biology, and flow rate.

Improvements:

1. Defined the SRB biofilm removal-based program.
2. Further refined our background.
3. Added hardware section and clarified the modeling scheme.
4. We decided to further search for background knowledge related to water supply and drainage systems, and conduct interviews with the engineering community, such as drainage groups and wastewater treatment plants.

May 17^th - Wastewater Utilization Specialists

Prof. Wenguo Wang is a specialist in the study of the utilization of wastewater resources from Chengdu Biogas Research Institute, Ministry of Agriculture and Rural Development. He has conducted research on quorum sensing of sewage microorganisms. Since the team members had no prior experience in studying biofilms, we hope we can get expert advice.

Problems:

1. E. coli is partially anaerobic, can it colonize in a sewer environment? Can it express sufficient, active enzymes?
2. How can biofilm samples be obtained from anaerobic cultures in sewers? What are the indicators for detecting biofilms?
3. The specific methods for detecting biofilms and quorum sensing, as well as the instrumentation for the test.

Opinions & helps:

1. The efficiency of E. coli AHL-degrading enzyme secreting is not high enough. Constructing a bioreactor that produces an AHL-degrading enzyme-sustaining agent, and then directly delivers AHL-degrading enzyme into the environment may be better.
2. It is feasible to obtain sewer biofilm samples by anaerobic culture, but it is difficult to obtain intact biofilm. However, biofilm validation can be done using existing biofilms in the sewers, which is closer to the real situation.
3. Affirms the option of culturing sewer biofilm samples through simulation experiments.
4. Provided data on biofilm composition in sewers and kept long-term collaboration on AHL and biofilm detecting experiments. We can detect polysaccharides and proteins in the biofilm for its integrity.
5. Further explained the biofilm composition of the sewer and reminded us of the re-living nature of free SRBs.

Improvements:

1. The program to build hardware for simulation experiments was further clarified.
2. Established a long-term cooperation with the Institute. Prof. Wang provides us with a platform for bioassays.
3. Provide substantive recommendations for biofilm and AHL assays.
4. Determine the necessity of using antimicrobial peptides to remove free SRBs.

Updated solution ver.5

1. Added the hardware part. We expect to simulate the environment of the drainage pipe in the laboratory and carry out the cultivation, and function verification experiment of the engineered bacteria.
2. Application: Considering embedding and placing the bacterial powder into the sewer. Select appropriate parameters for modeling based on the basic structure of the drainage system, biology, and flow rate.
3. Since directly constructing a bioreactor to produce AHL-degrading enzymes loses other properties of live bacterial therapy, we did not adopt this option.
4. Determined to add an antimicrobial peptide module, the treatment efficiency of the antimicrobial peptide was considered.

Integrate different insights from stakeholders

Jul.-Aug. 2023

Jul. 10^th - CCiC

After visiting experts in academia, SRBioQuencher took an initial shape. In the CCiC conference, we listened to the official lectures as well as the speeches from various iGEM teams in China and exchanged the details of our topic with them. Meanwhile, we shared the design of our project at that time. We collected many valuable advice:

1. The introduction of an antimicrobial peptide module enhances the efficiency of the system.
2. Nature is also an important stakeholder. We should further investigate the biosafety issues when SRB biofilm is flushed into natural water bodies.
3. Further investigations are needed to highlight our advantages over traditional methods.
4. A regulatory system that uses hydrogen sulfide as a signaling molecule can be designed in E. coli. Concerned about the problem of hydrogen sulfide production in E. coli itself, we decided to use hydrogen sulfide conversion to remove them. and expected to increase the removal of hydrogen sulfide in the cocktail therapy. A section expressing SQR protein (thioquinone oxidoreductase) was added.

Jul. 16^th - Prof. Yongzhen Xia

In the pre-experiment, the team had problems with heterologous expression of thioketone oxidoreductase (SQR). We consulted Prof. Yongzhen Xia from Shandong University. He provided us with the protocol for the purification of SQR membrane fractions in previous work and the SQR sequence from Cupriavidus pinatubonensis JMP134. helped the experimental work.

Jul. 20^th - Beijing Drainage Group CO., LTD

During the summer, we visited the operation and maintenance departments of drainage systems in different parts of China. While gaining background knowledge about drainage systems, we also continued to gather views from experienced engineering application specialists on our project.

In Beijing Drainage Group, we talked with Zhao Zhendong and Wang Miao, engineers in charge of drainage pipe maintenance. The experts first confirmed the significance of the project and pointed out that H₂S and CH₄ are indeed the two main focuses of current scientific research on drain gases, with H₂S being the most prevalent. The treatment of biofilm in sewers, is a more popular research direction and a future trend in drain maintenance.

Problems:

1. Is the environment in the drain anaerobic? What are the approximate characteristics of effluent pH, bacteria, and gas distribution?
2. What is the ideal solution for engineering bacteria placement?
3. Will putting engineered bacteria into the drainage pipe cause biosafety problems?

Opinions & helps:

1. The closed structure and the full pipe flow in the drainage pipe can provide an anoxic environment for bacterial growth. Outlet pipes and primary pipe areas, and areas where pipes have special structures (corners, sinks, etc.) are usually measured with higher H₂S concentrations. Effluent pH is strongly influenced by upstream.
2. Direct placement of engineered bacteria is a better option. Calculations are needed to determine the distance and time interval between the placement points (considering pipe diameter, flow rate, etc.). At the same time, the area with more SRBs should be focused.
3. The number of E. coli is not a criterion for monitoring the environment of the drainage line. Most of the E. coli are removed during the wastewater treatment process. Of course, a small amount of engineered E. coli may survive and be discharged into natural water bodies and require attention.

Improvements:

1. Refine the idea of the release model.
2. Determine to direct release of engineered bacteria and conceptualize the hardware to be used for release and monitoring.
3. Rethinking the biosecurity issue and adjusting the suicide mechanism and the release program to reduce the possibility of engineered E. coli leaking into the natural environment.

Aug. 4^th - Chengdu Xingrong Drainage Group

At Chengdu Xingrong Drainage Group, we learned from Mr. Ouyang Houjie and Ran about the local drainage system and the main problems it faces. They introduced the national standards that need to be complied with during the operation and maintenance of the drainage system.

Opinions & helps:

1. Concern whether the addition of engineered bacteria will influence the biochemical reaction in sewage treatment.
2. In terms of application, the high flow rate of the main sewer pipe and the installation of the placement device directly in the pipe might be washed away by the water flow and the engineered bacteria would be quickly diluted.

Improvements:

1. Decided to contact the wastewater treatment company to ask about the composition of the effluent and whether the engineered bacteria would affect the biochemical reaction.
2. Rethinking the placement strategy and deciding to place the engineered bacteria mainly in the upstream and midstream branches to address the root cause of the problems that arose.
3. The provided level change charts and flow rate simulations provided data for the placement process, and various national standards also provided references for the modeling work.

Aug. 11^th - Chengdu No.4 Wastewater Treatment Plant

Following Mr. Ran's questions, we contacted Chengdu No. 4 Wastewater Treatment Plant. Engineer Zhang Zongyi introduced the process of sewage treatment and told us that the treatment plant conducts daily water quality monitoring of the sewage to determine whether it meets treatment and discharge standards.

Problems:

1. The input of engineered E. coli may cause changes in the levels of compounds in the sewage, will this affect some of the biochemical reactions in the treatment of the WWTP? Will it affect the pH of the influent?
2. Will our anaerobic engineered E. coli be able to be treated cleanly at the WWTP?

Opinions & helps:

1. For sewage treatment plant, the optimum value of the compounds in sewage is C: N: P=100:5:1, but a certain degree of fluctuation will not affect the treatment, and those that do not meet the standard can be exogenously placed. The pH of the influent water is fine if it is between 6-9.
2. The aerobic tank in the plant is artificially fed with a large amount of oxygen, which removes most of the bacteria. The rest is removed in the ultraviolet sterilization tank.
3. The main component of the sludge in the sewage plant is a variety of microorganisms for water purification, and it is necessary to ensure that the engineering bacteria will not affect the microbial function of the sludge.

Improvements:

1. The range of impacts of controlling engineered bacteria on the wastewater treatment system was clarified, and the criteria were not difficult to meet.
2. Understood the criteria for sewage, discharge back to treated water in natural ecosystems.
3. Focuses on the effect of engineered bacteria on bacteria in activated sludge.

Updated solution ver.6

1. The application of the project was clarified: direct embedding and delivery of engineered bacteria powder. The basic structure, biomass, and flow rate of the drainage system were modeled based on real data and national standards.
2. Further clarified the consideration of GMO leakage problem in the process of engineered bacteria placement after understanding the process of wastewater treatment and national standards. We designed a suicide device and validated its results.
3. Conducted surveys and interviews outside of the laboratory with workers and urban residents, two of the groups most susceptible to the effects of drain gases, in hopes of making the topic beneficial to society on a larger scale.

Jul.-Aug. 2023 - iGEM Teams

SCU-China values input from other teams in the iGEM Community. We actively communicate with teams that are geographically and subject matter similar. We participate in and invite other teams to participate in inter-collegiate exchanges and continue to grow from the input of our peers.

1. NEU-China: NEU-China is a team focused on the research of biofilm synthesizing. They instructed us not to forget the biofilm formed by E. coli itself, which may affect the system.
2. HainanU-China: This year, HainanU-China is working on the application of antimicrobial peptides in acne treatment. We mainly talked about the problem of antimicrobial peptide screening and the detail of putting forward wet lab works. They also inferred the infection of engineered E. coli in the environment.
3. UESTC-China: UESTC-China and SCU-China are both located in Chengdu City. We both think regional cooperation has a special status in the iGEM Community. The teams shared their project detail with each other, and shared local resources and cooperation opportunities in Chengdu.

Explore the meaning and connect the world

Sept. 2023

After refining the theoretical line and applied methodology of the topic, we began to explore the values of SRBioQuencher in the Sustainable Development Goals of the United Nations and bioethics. In the exploration of social values and humanistic issues, more improvement comes from the emergence of a collision of ideas and inspiration in the process.

Jul. 27^th - iGEMer Speaks

At the invitation of UESTC-China, SCU-China participated in the iGEMer Talk activity together with WHU-China NPU-China, and ZJU-China. We mainly opened a conversation on Human Practices and Sustainable Development Goals.

Opinions & helps:

1. Human Practice can be fed into the work of the humanities. For example, it can be used to provide advice on problems identified in the course of the work or to publicize science to the target group.
2. In the study of the Sustainable Development Goals (SDGs), we can try to contact regional UN offices for cooperation, exchange, and help.

Sept. 18^th - Synthetic Biology x Sustainable Development Conference

September 18th, SCU-China participated in the Synthetic Biology x Sustainable Development Conference held by team Tsinghua-TFL. At the conference, all 6 teams participated and talked about how SDGs inspire their iGEM projects.

Opinions & helps:

1. The exploration of environmentally based Sustainable Development should focus not only on the environment itself but also on the social and economic impacts of environmental problems and solutions.
2. The focus of the exploration of sustainable development should be contextualized.

September - Collaborate with McGill iGEM on Bioethics

In September, we collaborated with McGill iGEM on a Bioethics Report. Thanks to the chance, we analyzed our project according to McGill’s guide on how to consider an environment-themed project on bioethics. We considered 4 perspectives: biosafety, biocontainment, pollution and use of natural resources, and socio-economic impacts.

This is the environment chapter of the original report. Our section is on page 9.

Updated solution ver.7

1. After conducting surveys and interviews, we plan to produce the interviews in the form of videos and posters to engage more residents to protect the sewer system.
2. In the process of Human Practice's documentation and analysis, we need to be more adapted to the local environment and economic conditions of Chengdu.
3. Try to get in touch with the United Nations to explore more ways to expand the humanistic meaning of the topic.

Realizing our limitation

At last, we reflect on the limitations of our work of Human Practices. We expect to gain insights and progress from more after iGEM exchanges and reflections.
The effectiveness of SRBioQuencher:
Due to schedule constraints, our experiments with engineered bacteria were limited to the lab only and lacked the desired quantitative results. Although we predicted the de-disposal effect and economic impact of SRBioQuencher through modeling, more reliable experimental results are needed if we want to increase stakeholders' willingness to invest in the product and gain general public's trust.
Biosafety:
The application of engineered bacterial products in environmental management, especially in infrastructure maintenance, remains in the laboratory. As a result, relevant national standards point more to the environmental pollution caused by the native bacteria in the system than to the biosafety risks of GMOs. Therefore, the industrial community stakeholders we interviewed did not express very sharp skepticism about this.
We are concerned that even if we design an efficient suicide system and confirm that E. coli can be removed from a wastewater treatment plant, there will still be a small number of engineered bacteria leaking into the natural environment in practical applications. Subsequently, this issue needs to be discussed with GMO safety experts.
Socio-economic restriction:
In our country, the operation and management of the drainage system is the responsibility of state-owned enterprises. In the routine operation and maintenance of the drainage system, the enterprises must consider the cost in addition to ensuring the effectiveness of repairing the damage. Therefore, although there are a lot of efficient solutions for SRB biofilm under research, they have not stood out and gone into application. The best practice to reduce the cost is to breed strains that are both stable and effective and to design a viable application program for it. But until then, we have a long way to go to get enough investment to start the business.