Our project aims to engineer the cell factory using synthetic biology for 1,5-pentanediol (1,5-PDO) bioproduction. 1,5-PDO as a material monomer can be widely utilized in various fields of modern society, such as paints and coatings. We hope using cell factory to produce 1,5-PDO from renewable raw materials to contribute to biomanufacturing and green sustainable development. To better complete our work and achieve our objective, a series of Human Practice activities have been carried out. We first conducted a brainstorming session to discuss the project stakeholders, implementation plans, and methods. We consulted with large-scale enterprises, startups, researchers, and doctoral candidate to understand what society brings us, how we can improve the program, and how our programs impact society.
The entire Human Practice section is cohesive, closely connected to society, and is responsible for the world.

To make sure our project is responsible for the world, we held a brainstorming session to discuss a series of problems. Based on the discussion results, the project's purpose, project's stakeholders, implementation palns, and project's influence were analyzed in detail respectively, including priority level, and introduction in a mind map of the project. With this as a guide, we implemented our human practice and adjusted them to fit the actualsituation.

Figure 1 Brainstorming Session

1.Which resources or communities do you consult to ensure those are appropriate values in the context of your project?
a.Masses
b.Researchers in Synthetic Biology
c.Industry professionals
d.Government regulations

Figure 2 collation analysis diagram(Q1)

2.What evidence do you have to show that your project is responsible and good for the world?
a.Feedback and suggestions through interview and communication activities
b.Questionnaires
c.Brochure
d.Public acceptance and Environmental Assessment

Figure 3 collation analysis diagram(Q2)

3.What impact will your project have?
a.Environment
b.Sustainable development
c.Bio-manufacturing and synthetic biology research
d.Human health
e.Education
f.Market

4.Who are your proposed end users?
1,5-PDO, a material monomer, is widely used in the manufacturing of products such as polyurethanes, polyesters, plasticizers, inkjet inks, coatings, and fragrances. Therefore, our end users include all people related to material monomer manufacturers and manufacturers using 1,5-PDO as raw materials.

5.How do you envision others using your project?
a.Coating-related companies
b.Biobased materialsc
c.Environmental organizations
d.Research institutes
e.Research teams
f.Startups

6.How do you implement your project in the real world?
a.Project design
b.Project optimization
c.Project promotion
d.Project implementation
e.Project landing

Through the brainstorming session, we worked out our plan suited our project. Based on this, we carried out our Human Practices activities in three parts and formed “3 cycles” Loop unique design.

Figure 4 Summary of brainstorming ideas

With the rapid development of science and technology, carbon emissions have also increased. Carbon reduction has become an urgent goal for "all of humanity." Many countries and organizations have also promulgated relevant measures.
In 2015, the United Nations adopted the 2030 Agenda for Sustainable Development, an action plan covering 17 Sustainable Development Goals (SDGS).
The United States government signed Advancing Biotechnology and Biomanufacturing Innovation for a Sustainable, Safe, and Secure American Bioeconomy in September 2022, launching the $2 billion National Biotechnology and Biomanufacturing Initiative.
In 2018, the EU updated its bioeconomy strategy, proposing to accelerate the deployment of a sustainable European bioeconomy to contribute to the achievement of the Sustainable Development Goals and the target of the Paris Agreement.
The 14th Five-Year Plan for the development of the bio-economy proposes the substitution of traditional chemical raw materials with bio-based materials, the replacement of traditional chemical processes with biotechnology, an 18% reduction in carbon dioxide emissions, and the development of green and low-carbon biomass alternative applications. In the World Economic Forum's "Davos Agenda," China proposed to strive to peak carbon dioxide emissions before 2030 and achieve carbon neutrality before 2060.
The support of the policy provides a solid guarantee for our team to carry out the project, so that we can play the creativity.

Sinopec Beijing Research Institution Of Chemical Industry.
Sinopec Beijing Research Institute of Chemical Industry, a scientific research institute directly under Sinopec, is the largest research institution engaged in comprehensive petrochemical research in China. Sinopec's experts fully affirmed the potential of bio-based materials.
In recent years, they have also looked at bio-based materials and received positive feedback. The experts mentioned that due to the country's policy of "carbon peaking and carbon neutrality," future production will inevitably prioritize environmental protection and sustainable development. Petroleum-based raw materials, with their inherent limitations, will lead to an increased focus on products from renewable raw materials by bio-manufacturing and biosynthesis. Some biosynthetic products, such as bioethanol and bioplastics, show good momentum and are expected to gradually replace certain petroleum-based products in the future.
Furthermore, experts provided recommendations for our project. Two crucial points should be considered. Firstly, it is essential to translate theory into practice to advance the maturity of biosynthesis methods. Secondly, it is vital to bridge the gap between biosynthesis and the industry, successfully achieving industrialization. The production of bio-based materials at a similar or lower cost compared to traditional petroleum-based and coal-based materials will undoubtedly gain a significant market share and have a promising market outlook.

Figure 5 Discussion with Sinopec's experts

In the process of communicating with experts, we learned about the prospect and feasibility of biosynthesis and biomanufacturing. Bio-based products have the potential to align well with the national "carbon peaking and carbon neutrality" policy. Through the exchange, we are firm in our project objective, utilizing synthetic biology to engineer cell factories for biosynthesis. In addition, we are well aware that biosynthesis still faces numerous problems and challenges before it can be fully developed for large-scale industrial applications. We still have a significant amount of work to do if we intend to implement the project.

Through discussions with experts, we gained a clearer understanding of the policy, status and prospect of bio-manufacturing and bio-based products. They served as a crucial foundation for the project's core principles, making us firmly set the project objective.

Figure 6 Group photo with Sinopec experts

It is a company with outstanding achievements in the field of coating and marine industry protection technology.
Director Weimin Tan mentioned that the scope of application of bio-based material monomers in coatings has been increasing in recent years, which has been widely concerned by petrochemical companies such as CNOOC. Some products, such as the curing agent prepared from bio-based material monomer 1, 5-cadaverine, have entered the market.
We further consulted Director Weimin Tan about our target 1,5-PDO. We learned that 1,5-PDO with hydroxyl groups has a wide range of applicationsin the coating industry, such as diol polymerization of polyester, polyurethane, and other materials. With the addition of 1,5-PDO, the polyester, polyurethane, and other coatings exhibit improved performance, such as wear resistance, gloss, thermal stability, and adhesion. It also found to be applied in high-end fields such as aviation and in the life market. In addition, Director Weimin Tan also suggested that the shift from petroleum-based material monomers to bio-based material monomers is an appealing aspect for businesses, but it is crucial to identify the specific advantages.

Figure 7 Visit to CNOOC Research Institute

Figure 8 Conversing with a CNOOC’s expert

During our discussions with Director Weimin Tan, we learned about the bio-based material monomers and the advantages of polyester and polyurethane coatings produced from 1,5-PDO. Combined with the recommendations, we carried out a large number of investigations to clarify our target of the project: bio-base 1,5-PDO.

Throughout the discussions with Director Weimin Tan, we recognized the vast potential of bio-based material monomers and identified the biosynthesis of 1,5-PDO as the target product of our project.

Through research and consultation on national policies and social enterprises (Sinopec's experts and CNOOC's expert), our team has clarified our project: to biosynthesize 1,5-PDO, bidding farewell to traditional petroleum-based raw materials and conventional chemical synthesis. Our goal is adopting biomanufacturing by engineering a cell factory for the production of 1,5-PDO from renewable carbon source.

Professor Bingzhi Li, has extensive experience in participating in the iGEM competition. His research mainly focuses on synthetic biology, DNA information storage, and biomass conversion.
During the exchange, we consulted with Professor Li about the overall design of functional modules and the metabolic pathway for 1,5-PDO biosynthesis from glucose. Professor Li approved our modular design of metabolic pathway in synthetic biology and provided relevant suggestion. Additionally, he pointed out that our promotional materials were not vivid enough and suggested to add illustrations or diagrams to enhance comprehension.

During the communication with Professor Li, the metabolic modules of 5-HV synthesis module and 1,5-PDO synthesis module designed by us were recognized. Therefore, we conducted follow-up experiments based on this metabolic pathway.

During the communication with Professor Li，We determine our modular design of metabolic pathway in synthetic biology

Liya Liang is an associate professor in Dalian University of Technology. Her primary research focuses on the efficient preparation of bio-based products, particularly addressing key scientific issues, such as regulation of microbial metabolism and microbial catalytic engineering.
During our communication, Professor Liang highlighted that 1,5-PDO synthesis module may be a key module. However, the pACYCDuet-sfp-MmCAR-YahK contained a replicon with low copy number. She suggested replacing plasmids with higher copy numbers to enhance the expression of the 1,5-PDO synthesis module, potentially leading to 1,5-PDO yields.

After communicating with Professor Liang, We learnt that replace plasmids with higher copy numbers to enhance the expression of the 1,5-PDO synthesis module. So we decided to adopt a high-copy number plasmid to express the 1, 5-PDO synthesis module.

After communicating with professor Liang，We adopted a high-copy number plasmid of pRSFDuet-1 to express the 1,5-PDO synthesis module.

Figure 10 Interview with Professor Liang

Yingxiu Cao is an associate professor from Tianjin University primarily conducts research in the field of synthetic biology. Her research interests include genome design and synthesis, chromosome structural variation, functional remodeling, and intelligent DNA manufacturing.
In our discussion with Professor Cao, we primarily described the module design of 1,5-PDO biosynthetic pathway, including the lysine synthesis module, the 5-hydroxypentanoate synthesis module, and the 1,5-PDO synthesis module. Professor Cao encouraged us to adopt modular designing idea, and she suggested us to assess whether engineering of branched pathways could enhance the product titers. Furthermore, Professor Cao expressed strong interest in our iGEM project and reminded us to emphasize the style of our team. She emphasized the importance of creating a high-quality wiki and HP sections.

After communicating with Professor Cao, we learned that assess whether engineering of branched pathways could enhance the product titers. So we decided knockout of genes in the branched pathway to improve 1,5-PDO production.

After communicating with Professor Cao, we decided to construct two strains NT1003-ΔackA-pta and NT1003-ΔYcjQ by the CRISPR/Cas9.

Figure 11 Interview with Professor Cao

Xinkun Ren is an associate professor from Nanjing University. His team have focused on addressing the country's significant technical needs in recent years. They are actively expanding the application of new biological enzymes in energy, medicine, and the chemical industry, among other fields, achieving significant advancements in synthetic biology.
In view of the modification of key rate-limiting enzyme, he proposed. Professor Ren pointed out that random mutation is time-consuming. We could engineer of key rate-limiting enzyme of MmCAR by employing systematically designed methods to identify functional sites, and perform targeted mutations accordingly.

After communicating with professor Ren, we learnt that we could engineer the key rate-limiting enzyme of MmCAR by employing systematically designed methods to identify functional sites, and performed targeted mutations accordingly.

After communicating with professor Ren, we decided to carry out targeted mutagenesis of MmCAR by rational design and verified the effect of mutantion on its catalytic activity.

Figure 12 Interview with Professor Ren

Professor Jiaxing Xu from Huaiyin Normal University engages in research on enzyme catalysis.

In our communicating with Mr. Xu, we discovered that we can improve stability and catalytic efficiency by employing technical approaches such as optimization of catalytic conditions, enzyme modification, and enzyme assembly. Following a comprehensive literature review, our team members have decided to employ a strategy of enzyme assembly based on the protein scaffold to improve the synthesis efficiency in our project.

After communicating with professor Xu, we discovered that we can improve stability and catalytic efficiency by employing technical approaches such as optimization of catalytic conditions, enzyme modification, and enzyme assembly.

After communicating with professor Xu, our team members have decided to employ a strategy of enzyme assembly based on the protein scaffold to improve the synthesis efficiency in our project.

After determining our direction and objectives, we began the process of translating theory into practice. We received invaluable assistance from various experts throughout our project's lifecycle, ranging from the goal, the conceptualization of experimental idea, feasibility, implementation, improvement, and optimization. The involvement of various experts enriched our project with a multitude of perspectives and ideas, resulting in the healthy development of our project.
We integrated the opinions of people from all walks of life and experts into our practice. In the actual experiment operation process, we achieved the project improvement loop.

Figure 13 The project improvement loop

This company was founded by a doctoral candidate Wang Jing and her collaborative project team from Nanjing Tech university. Bio-based monomers are their main products. Her team works on the development of bio-based cadaverine and its derivatives, and successfully puts the company's technology and products on the market. Their achievements have been recognized with multiple awards in China College Students' “Internet+” Innovation and Entrepreneurship Competition.
Wang Jing, Liao Yang and Yang Yue provided suggestions on technology implementation and project promotion. First of all, we must develop bio-manufacturing technologies with competitive advantages and determine the maturity of our technology, which can find mature enterprises to cooperate. Under the precursors that guarantee technological maturity, project promotion required to comprehensively compare the advantages and disadvantages of competition through market analysis, industry background, identifying target customers, and seeking appropriate promotion strategies. The initial stage of introducing a new product is crucial as it marks the beginning of its entry into the market and establishes the foundation for future development. To expand market reasearch and enhance product awareness, preferential policies can be formulated, including offering free trials to initial downstream customers, providing free technical support, collaborating with downstream customers on product development, and offering flexible billing periods for high-quality customers.

Figure 14 Interview with Company Members

After considering these suggestions, we realized that the transition from technology development to start-up was a complex and comprehensive process. Mature technical support is the foundation, but equally important is, identifing our target customers, formulating appropriate promotion strategies, and gradually accumulating our customer base to expand the popularity of product technology.

To make synthetic biology, iGEM and our project are accessible to a broad variety of publics, we conducted a series of educational activities targeting different age groups, including kindergarten, primary school, middle school, university, and society. This deliberate approach ensured that we enable comprehensive efforts, gain valuable insights into the varying viewpoints and perspectives of different age groups regarding our project, and improved our project. Meanwhile, our efforts hoped to raise people's awareness about synthetic biology, biomanufacturing, and microbial cell factories, then made positive contributions to education and the sustainable development of society.

Figure 15 Education

See the Education page for details.

We mentored a high school team, actively engaged in collaborative activities with many teams and participated in numerous academic conferences, including the CCiC, Nanjing iGEM community, Environmental Protection and Sustainability Summit, The 15th Training Course of Coating Formulation Design and The 10th Training Course of Waterborne Technology, and The 7th National Annual Conference of Chemical Engineering and Biochemical Engineering. These provided us with insights into the various problem-solving approaches employed by researchers and different teams, pushing our project. We also met a captivating group of iGEMers and brought our support and advice to their projects.

Figure 16 Communication and Collaboration

See the Communication and Collaboration page for details.

Based on these activities, we have accomplished a unique design “3 cycles” loop.
We conducted a brainstorming session to formulate project stakeholders, implementation plans, and methods. To understand what society bring us, we researched national policies and consulted with Sinopec's experts and the CNOOC's experts, clarifing our project. To understand how we can improve the program, we received invaluable assistance from various experts throughout our project's lifecycle to escort the sound development of our project. To understand how our programs impact society, we consulted with startups to learn about the process from technology development to start-up, conducted a series of educational activities, collaborative activities with many teams and participated in numerous academic conferences. It begins with the thoughts and challenges presented to us by society, then explores how the relevant individuals can enhance our projects, and concludes with our own contributions to the advancement of society. The entire Human Practice section is cohesive, closely connected to society, and is responsible for the world.

Figure 17 “3 cycles” loop