Integrated Human Practices

Overview of the Problem

Water is one of the substances necessary for human existence and activities, especially the Chinese people. River culture has a special place in the history of China, with a special image and color. Chinese myths and stories, such as Yu Dayu's water control, are closely related to water sources. It has given the nation a sense of catastrophe, enjoyment, creativity, and imagination.

When it comes to Chinese people, the small-scale peasant economy[1] has relied on water resources since ancient times; Crops can thrive when it is irrigated. Even our parents' generation had childhood memories of playing in the water and helping the elders to irrigate the crops. We also played water fights in the river, fished, and immersed in the natural beauty in childhood;

Although the city is now prosperous, some rivers have become dirty and stench; in Xiangcheng District, Suzhou, where factories gather, residents there are complaining about the poor living environment, condemning the unscrupulous behavior of factories to discharge sewage.

Considering that environmental governance is against commercial laws, factories discharge sewage[2] into rivers and lakes in order to save costs, causing serious water and soil pollution. Therefore, guided by the low cost of products, we seek to use biofilm as a method of turning waste into treasure to contribute to metal ion water pollution.

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Serious Harm of Metal Ions to Water and Soil

According to the survey[3], more than 80% of rivers, lakes, and oceans in China face serious pollution problems from heavy metals,especially Cd, Hg, and Pb. In the electroplating industry, metal rinsing, mining, and heavy metal-polluted wastewater into the environment, threatening human health and the ecosystem(water and soil). Heavy metals are not biodegradable and can cause serious health problems for organisms.

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Discover the Problem: field interview

Suzhou is located in the middle and lower reaches of the Yangtze River in China, east of Taihu Lake.[4] People here have lived by rivers since ancient times. The water and land double chessboard pattern of Suzhou gardens is a national civilization. Therefore, starting from the local characteristics, we pay special attention to the protection of water resources.
In March, we have noticed a WeChat tweet reporting a case of excessive sewage discharge. The content of the tweet is as follows:
' Before the epidemic, the Green Jiangnan Public Welfare Organization sampled the water source at the location of the an electronic discharge pipe and sent it to an institution for testing. According to the test results, the copper ion concentration in the water sample reached 58600ug/L, and the copper ion effluent standard is 300ug/L, nearly 200 times higher than the standard. '

Then, we went to visit the non-profit organization Green Jiangnan. In the course of our discussions with this charity, we learned that heavy metal pollution in industrial wastewater is still a pervasive problem. However, studies have shown that due to their non-degradable nature, heavy metals can pose serious ecological and biological health risks when they enter the environment.

We talked about the heavy metal pollution problem with Green Jiangnan. The director Mrs. Chao suggested we should first investigate the existing water purification methods that are now commercially available to treat heavy metal ions. Then, we can design our products through synthetic biology.

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Discover the Problem: literature Review

After asking Environmental Technology Ltd and reading government published industry reports, we found that the current wastewater treatment methods are the traditional chemical precipitation and ion exchange methods. In addition, membrane filtration methods can also be included in the treatment of water. The initial communication with the stakeholders provided the basic framework for our further understanding.
We learned about the characteristics,advantages and disadvantages of existing wastewater treatment technologies through literature review. We found that most of the options they have chosen produce secondary chemical pollution due to the cost control of industrial production. If our product can reduce environmental pollution while ensuring purification efficiency and can maintain or reduce the price, it will be market competitive. To ensure the necessity and competitiveness of the product we design, we need to understand current market trends and existing competitors. Therefore, we planned a site study of a sewage plant.

Principle of Integrated Human Practices

Referring to the excellent wikis of previous teams, and asking XJTLU-CHINA's seniors and instructors in previous years, we determined HP's research logic.

1. 1.Understand the Local problem
2. 2.Define a Good Solution
3. 3.Ideate our Solution
4. 4.Implementation & Evaluation

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Part1 Understand The Local Problem

Understanding the problem of sewage treatment is the foundation of the research problem. From the perspective of stakeholders, we communicated with the community and discussed the issue of sewage treatment.
The stakeholders are as follow:

1. 1.Surveillance authorities: Environmental protection departments, Pollution detection companies, and Non-profit organizations;
2. 2.Sewage enterprises: Electronics Factory, Capacitor Electric Company, and Copper strip cleaning factory;
3. 3.Sewage plants： Domestic and Industrial Sewage Treatment Plant；
4. 4.Public: People of different ages and professions

Stakeholder 1: Surveillance authorities - Pollution detection companies

The literature review gave us a deeper understanding of the problem, and the wonderful application and superior function of biofilms in the literature also gave us full confidence. Therefore, we tried to use synthetic biology[5] to design our experimental device from the perspective of market value and cost reduction.

We would like to have a more in-depth understanding of the problems of sewage treatment in Suzhou from a technical level.We visited Shanghai Cheng Feng Technology Corporation which is a company for water quality testing. They cooperate with the supervision department a lot and have a good understanding of the local problem in Suzhou. The chairman Mr. Zhao showed us the method for water quality testing.

We should focus on the following main components:cost, removal efficiency, and pollution. We have to communicate with different industries and collect the wastewater samples from them so that we can measure the real heavy metal ion content in the wastewater.

Stakeholder 2: Sewage enterprises- Electronics Factory

For the engineering bacteria in our product to have future potential for use, we needed to first know the concentration of heavy metal ions in untreated sewage in real-life situations in order to know whether our engineered bacteria could be adapted to the relevant concentrations or whether some adaptations were needed. 
After a brief introduction to our project with a technician from Fuwei Technology (Wujiang) Co Ltd, he sent us a sample of water before treatment. At the same time, we went to an electronics factory for on-site sampling. This will help us analyze the pre-treatment effluent data very well and the concentration of heavy metal ions. This made our subsequent communication with the stakeholders more convincing. 
Having obtained the water sample, we could better decide the marketing position of our purification product in the wastewater treatment procedure. We also shared the water sample with peking and helped them conduct their project.

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Stakeholder 3: Sewage plant- Industrial Sewage Treatment Plant

Walking through the sewage plant in the new district of Suzhou, we were given a tour of the entire process of sewage treatment at the plant. We were astounded by the huge efficiency of treating wastewater in industrial production mode and consequently felt the huge gap between the ideal model we wanted to design and the real-life application. Finding ways to bridge the gap between laboratory products and mass-produced equipment on an industrial scale will therefore be the focus of our hp work on this theme.

We talked to the director Mr. Zhu. He pointed out that the key factors of wastewater treatment are cost and filtration rate. He also showed us the principle of the AAO system, which helps us better understand the wastewater treatment procedures in practice. That also gives us ideas about the hardware design.

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Stakeholder 4: The public- Questionnaire & public engagement

The most important thing in aligning with real life is to understand the market needs and feedback from the public. Therefore, we have designed a questionnaire survey, focusing on the demands and public attitude to synthetic biology. Considering that synthetic biology, which we are about to use, is more cutting-edge technology, it involves techniques such as gene editing that may cause social controversy. Therefore, before designing it, we felt it was necessary to understand the level of understanding and acceptance of synthetic biology by the public. To collect more realistic and diverse data, we designed questionnaires that can be filled out online.

We received 473 questionnaires from the public. From the statistics, we found out that heavy metal pollution always occurs, which is enough to raise public attention. Thus, we can decide the demand for wastewater treatment. However, more than 90 per cent of the samples know little about the wastewater effluent standard. Moreover, we surprisingly found out that more than three fourth of the participants advocate the engineering biofilm adsorption method. Some people were still worried about biosafety issues and offered us lots of valuable suggestions.

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Part2 Define a Good Solution

We had a clear understanding of the problem after visiting these stakeholders. Then, we designed our project and device based on its application in the real world. Our experiments, modeling and hardware are designed to fully integrated with real-world needs and optimize the efficiency.

1. 1.Experimental materials: Biofilms contain the idea of turning waste into treasures; biofilms that are harmful to humans are converted into materials for sewage adsorption.
2. 2.Experiment and hardware design: It is highly integrated with the actual situation, using real sewage to design experiments. We adjust the hardware device in combination with effluent standards.
3. 3.Modeling: Modeling assists the experiment to verify the experimental results, and they iterate each other.

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Understand experimental materials: Biofilm

Having identified the core element in our project as the curli system, the extracellular components of biofilm. We found lots of articles about the harmful aspects of biofilm, especially In the treatment of chronic wounds. In the lower limbs of patients with chronic infections such as diabetes, the bacterial biofilm acts as a protective film for the bacteria against the external drugs and also destroys the proteins needed for wound healing, making it difficult to heal the wound. we planned a face-to-face meeting with experts in this field to understand biofilm in practice.

Removal of the bacterial biofilm has been a major challenge in treating wounds in endocrine patients due to its inherent high adhesion and regenerative properties. Normally, during medical treatment, it is usually only possible to treat indirectly by controlling blood sugar and regulating blood microcirculation, and some direct treatments, such as electrochemical modalities, are rarely applied in actual medical treatment. However, we consider that the adhesion and adsorption capacity of biofilms can be utilized for the adsorption of metal ions.

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Experiment and hardware design: Build an idea

During the interaction, we gained a deeper understanding of bacterial biofilm's high adhesion, environmental resistance, and adsorption properties. This led to a greater certainty that these characteristics of the bacterial biofilm could be exploited to modify the engineered bacteria using synthetic biology, making full use of its advantages by altering its genetic coding. After more literature research and some enlightenment from previous years' iGEM projects, we finally decided on the core theme of the project, which is to construct a sewage adsorption device with autonomous growth capacity and to use curli fiber nanostructures in E. coli to autonomously synthesize biomaterials for engineered cells.

This is the first draft which reflects our design and proposal. We find out the bioreactor models from papers and make improvements. The biofilm is a main component of the filler, which can be utilized to adsorb heavy metal ions with lost cost and great efficiency.

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Modeling:

We strive to achieve full-system simulations ranging from molecules to pathways, to bacterial growth, and finally to device hydrodynamic modeling. For more information, please visit our Modeling page.

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Part3 Ideate our Solution

Discuss with experts

After two weeks of designing and brainstorming, we decided to design a new biofilm water purification device as our solution. To ensure that our device would not become less efficient by enhancing environmental protection, we came up with the idea of using synthetic biology methods to increase the active adsorption of metal ions by engineered bacteria. However, designing a device was new to our group and therefore spawned many questions. For example, how would the device be built? How to fix our engineered bacteria? Will the rate of self-renewal of our engineered bacteria be balanced with the rate of death due to heavy metal ions?

Therefore, based on these questions, we plan to interview experts for their advice or ideas in the next step. After the group meeting, we collated the group members' doubts and grouped them into a set of questions, which mainly included the following.

1. 1.Engineered bacteria and fixtures fixation issues
2. 2.Questions on the design and construction of water purification units
3. 3.The question of whether it is possible to interface with real-life sewage
4. 4.The rest of the advice from experts...

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Prototype design: Discuss with Prof. Chen and Dr. Hu

Prof. Chen is an associate Professor in Xi'an Jiaotong-Liverpool University. He is an environmental chemist and fascinated by the microbes mediated extracellular electron transfer. We briefly described our project to Prof. Chen. Then, he gave us suggestions for device design, safety and experimental design.

Firstly, when it comes to device design, He pointed out that plastic and honeycomb form carriers may be a good choice for nutrients and biofilm adhesion. Then, he led us to a significant biosafety problem that is engineering bacteria leakage. He said that in our project design, the leakage of bacteria can cause serious safety problems. Last but not least, Prof. Chen also recommended that ICP (Inductively Coupled Plasma Emission Spectrometer) can be utilized for the detection of heavy metal ions.

He also kindly explain the principles of the project to us and answered many of our technical questions. For example, why gold and silver ions are easily reduced. We are very grateful to Prof. Chen for his great help, which made us have a better understanding of biological and environmental science

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Mr. Chen recommended Dr. Hu from the Chinese Academy of Sciences to us, and we exchanged questions about the design of the hardware with Dr. Hu.

Dr. Hu gave us a detailed introduction to the working principle of MBBR. There are two key points:1. Incubation period of the reactor; 2. Membrane flux range setting.

We had a pleasant exchange with Dr. Hu. We learned about the related problems that will arise in the design of the device and communicated with us about the environment-related issues.

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Experimental design and modeling: Discuss with Prof. Raju and Dr. Xu

We had a meeting with Prof. Raju, an microbial environmentalist in XJTLU, for suggestions on experimental design and verification. Raju gave us suggestions on biofilm measurement and safety concerns.

• 1.E. coli growth curve determination: how soon the E. coli can reach the stationary phase.
• 2.Experimental design: the control and comparison.
• 3.Sewage simulation: We can use synthetic water for measurement.
• 4.Safety ideas: See whether the plasmids of bacteria have environmental contamination and resistance; What if these genes into the environment?

We carefully considered the questions raised by Mr. Raju, redesigned the experimental protocol, and proposed our own solutions for safety issues. We evaluated the feasibility of the experimental operation to prevent biological leakage, and considered that maybe we could propose a variety of schemes, such as ultrafiltration membrane retention, suicide switch, and then choose whether to verify it according to the experimental process.

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We also discussed with Dr. Xu for advice on modeling. Dr. Xu instructs us to study the molecular mechanism and the kinetic pathway of the curli system. We communicated with Mr. Xu about the technical issues of modeling, and the accuracy and feasibility of whole-system modeling.

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Project concept: Discuss with Prof. Kappes

We had a meeting with Prof. Kappes, an associate professor of biology in DKU, for suggestions on Project conception.

Prof. Kappes first understood our experiments and pointed out a few areas for improvement: GST tag may influence E.coli growth, which means CsgA is close to GST tag so that it may fold incorrectly. When it comes to microscope, how could TEM and SEM handle biological sample, he recommended experts in relative experts. Then, he offered suggestions for human practices. Prof. Kappes reminded us to start thinking about the problem itself: whether it is an experiment or government supervision problem. He also stressed the importance of water source, which affects the land field, cereal, even harmful to human health. Lastly, he mentioned the safety problem and hardware design. The problem of plasmid transfer is very related to device design. We need to fully consider the shape and chemical properties of the filler, as well as the harm caused by the leakage of plasmids to the ecological environment.

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Hardware design: Discuss with Dr. Pow-Seng Yap

Regarding the hardware design, we communicated with teachers from the Department of Civil Engineering. Dr. Pow-Seng Yap is currently an Associate Professor in Xi’an Jiaotong-Liverpool University. He is also the Programme Director of MSc Sustainable Construction. He suggested that we design the installation with environmentally friendly materials, and recommended the seniors to ask questions and put forward his own opinions on the design of the installation.

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Part4 Implementation & Evaluation

Integrating Stakeholder and Expert Advice

We planned to utilize our product to replace the ion exchange or chemical precipitation method. Our product will not introduce other harmful chemicals into the water. As a result, we could use this biological method for refined adsorption.

Moreover, it was important to our team that we considered all technical, safety, ethical and socio-cultural concerns regarding implementation. To ensure this was achieved, our team decided to implement a two-phase project.

In Phase I , our team worked with the model organism, E. Coli, to solidify the foundations of our design. Phase II , will see this knowledge expanded in hardware design phase, when we apply these systems to real world by designing a bioreactor device.

Throughout our project, our journey has been entirely realistic, and it was no different for proposed implementation. When considering implementation, going back to our stakeholders and experts helped us close the loop, and ensured that the ethical, technical, safety[6] and communication decisions we made were informed by their needs.

To find out more about our proposed implementation, click Our Proposed Implementation.

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Closed-loop thinking: 3R criteria

• 1.Responsiveness: Communicate with society and get feedback from society;
• 2.Reflection: Reflect on the feedbacks from outside;
• 3.Responsibility: Take social responsibility and give back to society;

We find that the 3R principle officially given by igem is a closed loop of a good thinking process, which gives us a good logic and way of thinking and summarizing. Based on the principle, we communicate with society, reflect on the information from outside and give back to society.

In this part, we go out of school and come to the society to communicate with practitioners from all walks of life.

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Cycle 1: Technology or supervision?

Response from society: Communicate with entrepreneurs

We talked with Mr. Pang from Suzhou Huo Pu Environmental Protection Technology Co., ltd. We have interesting communication and acquired wider horizons from the entrepreneurs.
Mr. Pang suggests we investigate the commonly used methods for metal ion treatment again. Electrolysis, electrodialysis, and resin adsorption are also efficient methods for heavy metal treatment. He also kindly help us find out the market position of our product. It can be utilized in the last section for fine filtration.
However, after communicating with factories that recycle the heavy metal ions, we find out that nearly all of them use the chemical precipitation method for its low cost. Thus, the cost matters.

Reflection: Technology or supervision?

After communicating with practitioners in the society, we broke the concept of technology first, and really started from the problem to reflect on the social value of biological sewage treatment technology.

We used to attribute the water pollution problem to technology, but what really matters is supervision!
We called the environmental protection department several times but received no positive or valuable information about the sewage regulation. However, environmental protection non-profit organizations offered us a lot of methods for public monitoring and engagement. We will include this information in the education part.

Responsibility: How does the public engage in environmental monitoring?

For regulatory issues, we can draw attention to water pollution from the perspective of HP. Combined with education and publicity, we tell people how to monitor water quality issues, enhance the public's sense of social responsibility, and educate companies to reduce water pollutant emissions.

We can recommend the public to report the pollution issue in different ways, not just the environmental protection department. Some non-profit organizations can also be a good choice. Moreover, students who are interested in environmental protection can also engage in this kind of job.

Cycle 2: Biosafety issues

Response from society: Industry investigation

After finalizing the prototype of the product, we once again spoke to the another stakeholder. To ensure that our products are widely applicable to the market, this time we chose to interview the Executive Vice President of Jiangxi Eco-Dao Environmental Technology Development Cooperation.
During the interview, she showed a high level of appreciation for our products and thought our ideas were innovative and had practical application possibilities.

Mrs. Liu raised doubts about whether our gene-edited engineered bacteria could survive in a natural environment. For one thing, the plasmids are easily lost and the effect is lost; for another, the plasmids may drift to other microorganisms so that some environmentally uncontrollable mutations can occur. The genetically engineered bacteria may either not adapt to the natural ecology, or dominate the original ecosystem.

Whether engineered bacteria have an ecological niche in the natural environment is a question we have not considered before, and one that means whether our products can be produced in life. It is therefore the main objective of the next phase of our research, which is to make our products more marketable by gathering advice from experts in the relevant fields in society.

We connect with the factory and consider the actual value of this technology in practice.

Reflection: The vision and prospect - Three Visits to the Thatched Cottage

We have maintained close contact with Professor Chen. We visited this amiable teacher three times before, during and after the project. This was the third time we had visited Professor Chen of Environmental Science. Just like the traditional Chinese story: 'Three visits to the thatched cottage'. We esteem this helpful and kind-minded teacher and respectfully asked him many times.

When it comes to biosafety, Prof. Chen is lost in thought. Then a brilliant idea emerge. He not only provide another idea but future extension and outlook for our project.

Professor Chen put forward more possibilities and prospects for our project. Among them, the project prospect of bioming and bioleaching inspired us, maybe we can change our thinking and use the method of microbial oxidation of metal element to carry out tailings treatment, so as to reduce the harm of heavy metals to the environment.

• Biomineralization: Reduce the heavy metal ions

Biomineralisation[7] is the process of generating inorganic minerals by organisms through the regulation of biological macromolecules. In our experimental project designed to treat precious metals such as silver, biomineralisation helps bacterial biofilms to reduce the adhering metal ions to their nano form as monomers. This will provide favorable conditions for the subsequent recovery of precious metal ions. And recycling heavy metals can have the benefit of alleviating the paradox of resource scarcity in nature and reducing environmental pollution.

• Biomining & bioleaching: Oxidize heavy metal and extract the ions from sludge

Biomining[8] is the process of using microorganisms to extract the valuable minerals from ore in order to clean up sites with metal pollution and extracting rare earth elements.
It works when the microorganisms oxides the metals, which then allows the metal to dissolve in water and break away from the other solid minerals that are associated with. The ore containing the valuable minerals are placed in a heat pile. The bacterial solutions are applied to the heat pile in a process called heap leaching. Then, the oars placed in the agitator tank called vat that is shaken to speed up the bile leaking process.
Currently, biomining is used to produce 5% of the world's gold and 20% of the world's copper, but it can also be used to mind base metals such as nickel, zinc, cobalt and other rare earth elements.
The benefit of using biomining is that the microorganisms used are already found in the natural environment. Therefore, they post little to no environmental risk. Other benefits of biomining include low operating costs, low energy demand and low gaseous emissions.

Responsibility: Biosafety meeting

We held an online meeting with several teams on the 4th of September. We discussed about the biosafety issues together and the ideas will be compiled into a handbook for review.

Our main objectives are as follow:

• 1. Demonstrate that our experimental measures comply with safety rules
• 2. Prove that our gene-edited engineered bacteria will not cause leakage
• 3. Establish that our project will not cause harm to the environment or human health
  After reading all the requirements and regulations on the official website, we have set our safety objectives in accordance with the relevant standards.

In this meeting on biosecurity, we exchanged measures to deal with the situation according to the relevant requirements on the official website. The main measures are the design of suicide switches and the adoption of containment devices, which are the main ways in which we can deal with the leakage of engineered bacteria within the limits of our capabilities. In addition to this, we have also clarified the standardisation of laboratory practices and the handling of potentially harmful substances. Our solutions were also refined and optimised during the exchange.

During this session, we gained a deeper understanding of biosafety, so we also took notes on the parts of each varsity team's proposal that were worth noting, in the hope of providing some reference for subsequent competitions, which is another reason why we hosted the biosafety session - to leave some reference material for the upcoming competitions.