Overview

In this year, SZU-China innovatively created a new type of artificial sweetener production system. From the design of Biobricks to the establishment of a comprehensive legal network system to ensure biosafety, and to the creation of a rich world of sweetness, we have made extraordinary efforts in multiple aspects. Our team designed and characterized multiple new parts, including synthetic and storage system, and innovatively used the tomato fruit-specific promoter E8 and the vacuolar targeting peptide SPS-NTPP. To ensure a safer regulatory system for our genetically modified tomatoes, we explored the current management measures of various countries for genetically modified crops, and more professionally and specifically formulated a long-term supervision and safety guarantee manual for sweet proteins, ensuring food safety. In addition, we established a genetically modified team, promoted biosafety, and wrote a business plan. Finally, we recorded the difficulties we encountered in wet experiments and our problem-solving ideas, hoping to provide some suggestions for other teams who want to do plant synthetic biology.

Biobricks

We have constructed several BioBricks, ranging from synthetic modules to production modules. We have characterized them in detail in the laboratory and have documented the process.

Synthetic System

In the synthesis system, we divided the part into two parts: Prokaryotic Expression and Tomato Plant Expression.

I. Prokaryotic Expression

Ⅱ. Tomato Plant Expression

Storage System

In summary, by effectively constructing these BioBricks, we can stably and efficiently express the sweet protein Thaumatin in tomatoes, and by using a vacuolar signal peptide, we have accurately targeted Thaumatin to the vacuoles for storage, creating a novel sugar substitute production system. We have completed the experimental characterization of components such as E8 (BBa_K5160002), SPS-NTPP (BBa_K5160009), Thaumatin (BBa_K5160003) on the part page. For the characterization of the two components, Thaumatin (BBa_K5160003) and Brazzein (BBa_K5160004), we verified their expressions in Escherichia coli and tomatoes respectively. We have added the corresponding data and documents to the respective BioBricks, where we have detailed the origin, function, and how we characterized their use. Among them, E8 (BBa_K5160002), as a commonly used fruit-specific promoter in plant chassis, could bring great surprises to future teams attempting to work with plant chassis. Meanwhile, SPS-NTPP(BBa_K5160009), as a tool for vacuolar targeting, holds tremendous potential. In this vacuole localization verification experiment, we used and characterized the green fluorescent protein EGFP(BBa_K4251013) and supplemented its data. We hope that future iGEM teams get inspiration and enlightenment from our records and will enjoyably embark on their iGEM journey.

Learn more in Part.

In Food and Nutrition Village

The Biosecurity and Bioethics Whitepaper

The Biosecurity and Bioethics Whitepaper, which we participated in writing, aims to give more iGEM teams interested in genetically engineered foods a holistic view of the Food and Nutrition village as well as genetically engineered foods. We have written a series of content that introduces the Food and Nutrition Village in all aspects, project overview, village background, analysis of bioethics and safety laws and regulations, and discussion of biosafety measures. This contribution will enable future teams to understand the Food and Nutrition Village more quickly and start their projects with pleasure.

Meanwhile, SZU-China emphasizes the importance of responsibly producing synthetic biology foods in the The Biosecurity and Bioethics Whitepaper, highlighting the challenges faced by synthetic biology foods and exploring the urgent issues faced by the Food and Nutrition Village and digs into the pressing ethical issues that teams must consider. We hope that this initiative will promote a deeper understanding of synthetic biology foods among iGEM teams. Additionally, this report is an important shared educational resource (we have detailed this report on our wiki). In the future, any interested teams or the public can visit our wiki page to draw inspiration and aid in the formation of their projects. We also encourage more iGEM teams to join us, continually enrich this report so that we can foster communication and cooperation among teams to establish a vibrant iGEM community.

Fig 1. The Biosecurity and Bioethics Whitepaper

Robust Regulatory System

During our field research and visits, there is still a gap in the long-term regulatory system for food safety, which is why aspartame is still on the market even after safety problems have been reported. Although we visited the relevant departments and companies to learn about the appropriate precautions, the current regulatory system is still inadequate and difficult to ensure safety, not to mention food created using synthetic biology technology. However, food safety is a matter of human life and health. Therefore, for this reason, we have developed a more targeted and rationalized regulatory manual for food products produced by new technologies, the Genetically Modified Foods Long-term Safety Regulatory Manual.

By interpreting the regulatory measures of various countries, we have established a long-term regulatory system to safeguard food safety in our Genetically Modified Foods Long-term Safety Regulatory Manual, especially Thaumatin, which is favored by many people. We are committed to making food regulation transparent and visible^[4][5], which aligns with the iGEM safety spirit. We expect more iGEM teams in the food and nutrition village to join us in formulating their food safety policies, achieving diversified food regulation, and a cooperative and win-win iGEM community, establishing an integrated food safety protection network.

In summary, the safety regulatory system is dedicated to ensuring the long-term safety of food, so that it can respond quickly when harm arises, minimizing the damage to the public. Furthermore, we hope to establish an integrated global food regulatory system network, create more diverse and rich policies, and ensure food safety, especially for foods produced by new technologies. Together, let's build a promising future of food safety and scientific discovery!

Fig 2. Genetically Modified Foods Long-term Safety Regulatory Manual

Learn more in Integrated Human Practices.

Safety

Synthetic Biology Industry-Academia-Research Zone

The Synthetic Biology Industry-Academia-Research Zone of SZU-China is an excellent resource for the future transition of genetically modified (GM) crops from the laboratory to the test base and then to market promotion. Although the iGEM competition focuses on scientific innovation, ensuring that GM technology can safely enter society remains a significant challenge. Throughout the year, we have visited various institutions and read dozens of legal documents, deeply understanding the immense challenge of GM crops moving out of the laboratory. Therefore, we have established a GM group, possess our own Synthetic Biology Industry-Academia-Research Zone, and have obtained national approval for field trials of GM crops. This initiative undoubtedly provides theoretical guidance and practical opportunities for future iGEM teams interested in GM crops, solving the dilemma faced by other iGEM teams when dealing with GM, and saving time on researching GM crops. This is undoubtedly a valuable educational resource: teams from different professional backgrounds can learn about the details of GM crops moving out of the laboratory, receive customized and specialized guidance for Chinese teams, assist in their decision-making, and bring great surprises. At the same time, we hope that teams from different backgrounds will join us to establish a global GM team worldwide, promote the innovation of this technology, and thus benefit more people.

Fig 3. We are visiting the Synthetic Biology Industry-Academia-Research Zone

Our GM group and our own Synthetic Biology Industry-Academia-Research Zone are not only a theoretical educational resource but also an excellent natural "laboratory." In our Synthetic Biology Industry-Academia-Research Zone, we have obtained national approval for field trials of GM crops. This means that as long as GM crops are fully verified in the laboratory and the target genes are stably expressed, they can undergo field trials in the Industry-Academia-Research Zone. This is undoubtedly a great surprise for iGEM teams. It not only accelerates the pace of technology moving toward production but also provides a valuable natural laboratory for subsequent practices. In the future, the Synthetic Biology Industry-Academia-Research Zone will attract more iGEM teams, providing a platform and opportunities for their projects and promoting cooperation and exchange within the iGEM community.

Biosafety Promotion

In terms of safety, we not only ensure the safety of the project from multiple dimensions but also innovatively popularize safety knowledge to the public. Although our safety promotion is aimed at the public rather than a contribution to the iGEM community, we believe it is an extension of iGEM's social responsibility to the public. An excellent technology must come from the society and go into the society, which not only depends on excellent researchers and academics but also depends on the public's understanding, acceptance and utilization of the technology. An excellent iGEM program should be the same, the innovation of new technology will inevitably bring social concern and panic, but how to weigh this philosophical and ethical leverage requires us to take action.

Precisely because of this consideration, we spare no effort in promoting scientific knowledge of biosafety to the public, not only because biosafety is related to the interests and well-being of everyone, but also because we hope that the public can view the advantages and disadvantages brought by new technology more rationally, comprehensively, and dialectically. Through various approaches such as biosafety exhibitions, picture books, as well as laboratory safety videos, we have introduced biosafety to people of different ages and backgrounds, setting an example for other iGEM teams. Our goal is to extend outwards from iGEM, to radiate from international competition, and to encourage other iGEM teams to reduce the public's concerns about synthetic biology, establishing a rational and dialectical "synthetic biology" value system worldwide.

Fig 4. Sugar Substitute Science Picture Book.

Learn more in Safety.

In the Sweet World

Sweetness Detection

This year, SZU-China is planning to do something interesting, creating an innovative production system for a new type of sugar substitute, Thaumatin, using tomatoes as a chassis to create a richer world of sweetness. In the process of evaluating whether our genetically modified tomatoes have sweetness, we have created new methods for detecting sweetness from both the modeling and wet lab perspectives, abandoning the potentially risky human subjective sensory evaluation tests, ensuring the safety of genetically modified tomatoes. We unanimously believe that these contributions are of great significance as they provide new ideas for taste detection, avoid experimental errors and risks brought by subjective evaluation, provide direction for other iGEM teams, and create new perspectives within the iGEM community.

Model

This year, SZU-China has attempted to establish a molecular docking model of sweet proteins with sweet taste receptors in the modeling section. In the past, taste evaluation has always been tested through human sensory evaluation. This approach not only requires the pre-screening and training of volunteers but also professional venues and procedures, which are time-consuming, labor-intensive, and material-consuming. Moreover, it can be affected by human subjective feelings, thereby influencing the final experimental results. Therefore, to overcome the bias brought about by human subjective evaluation, we have explored the feasibility of using model to measure the perception of sweetness. In the modeling section, we have established a model of the binding of the sweet protein Thaumatin to the sweet taste receptors T1R2 and T1R3^[6]. By establishing a molecular docking model, we can investigate the effects of sweetness and the reasons for its persistence from a more rational and visual perspective, assessing taste perception from a new angle.

Fig 5. Schematic diagram of sweet protein Thaumatin binding to the sweet taste receptors T1R2 and T1R3.

At the same time, attempting to use models to solve taste evaluation issues is a significant breakthrough, providing new ideas and methods for other interested iGEM teams, thus explaining taste perception from different perspectives. We believe this contribution is immense, bringing us great surprises and promising to establish new sensory evaluation standards in the future.

See more in Model.

Wet Lab

At the same time, SZU-China also verified the sweetness of GM tomatoes by ELISA^[9] and electronic tongue^[7] in the wet lab section. This avoids the hazards and risks associated with the use of human sensory tests in iGEM events and provides a new method for sweetness detection.

Learn in more in Wet lab.

In summary, we have employed three different methods from two perspectives to fully verify whether our genetically modified tomatoes possess sweetness. This contribution is immense. It provides new ideas, perspectives, and approaches for sensory evaluation, offering a more convenient and faster detection method. This helps avoid the subjective biases of human sensory evaluation and prevents any potential harm to volunteers from tasting genetically modified tomatoes. By doing so, we can comply with the iGEM competition's requirements regarding human testing and become responsible engineers or scientists. Furthermore, this work is inspiring. Any interested iGEM team can not only learn from our methods but also gain new inspiration from them, applying these insights to their projects and bringing great surprises to the iGEM community and the world. Lastly, this work is innovative. We have delved deeply into the model and ELISA, drawing inspiration from them and endowing the model and ELISA with new functions and meanings, taking a significant step forward in innovation.

Hardware

This year, SZU-China has designed and constructed an automated detection device for common sugars based on lanthanide metal ester fluorescence. By utilizing the fluorescence changes of lanthanide metal ester fluorescent materials in different liquid environments, combined with Principal Component Analysis (PCA), the device achieves the detection of various common sugar contents. Compared to traditional sugar measurement instruments, our device is more sensitive and rapid, and can be applied to various settings such as laboratories and regulatory departments, enabling precise detection of multiple common sugars.

Fig 6. The usage procedure of the automated detection device for common sugars based on lanthanide metal ester fluorescence.

In addition, we have innovatively developed a rapid quantitative lateral flow immunoassay (LFIA) test strip for the Thaumatin protein. By using the common HA antibody target to engineer the protein expressed in plants with an HA tag, we achieve rapid quantitative detection of Thaumatin content in samples. Compared to traditional detection methods—such as Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA)—the method we've developed undoubtedly reduces costs, lightens the workload, and provides an economical, efficient, user-friendly, and scalable solution, thereby enhancing the feasibility and efficiency of screening.

Fig 7. Rapid quantitative lateral flow immunoassay (LFIA) test strip for Thaumatin protein.

The contribution of this work is also extraordinary. From a hardware perspective, we have laid the foundation for the detection in the world of sweetness. These devices offer an economical, efficient, user-friendly, and scalable solution for sweetness detection, thereby aiding in the establishment of a world of sweetness. Other interested teams can also understand the current market demand and influence of sugar substitutes.

Learn more in Hardware.

Business Plan

This year, in order to successfully launch our sweet protein and make it available to the public, we have proposed a business plan for the sweet protein based on the content of our team project. Our business plan includes a series of considerations that we took into account when writing the plan. We started with the unmet needs in the current market, conducted an in-depth investigation of social demands, considered the potential users in the market, and listened to their desires. We then explored the possibilities of the sweet protein entering the market from the aspects of technical feasibility, economic feasibility, and community feasibility, and finally, we formulated a development plan and estimated the future development opportunities and challenges. In addition to SZU-China's use of the data, other interested teams can also understand the current market demand and influence of sugar substitutes.

At the same time, our business plan is also a valuable educational resource. In entrepreneurship, we detailed the thought process behind writing this business plan and the ethical issues we considered. Other iGEM teams in the iGEM community can visit our wiki page to draw inspiration from our work, continuously optimize, and potentially promote cooperation between different iGEM teams in the future. We look forward to truly establishing a world of sweetness and bringing a great surprise to the world.

Learn more in Entrepreneurship.

Connection

SZU-China is committed to establishing deeper friendships with other iGEM community members, building bridges for communication and cooperation, and expanding our influence beyond Shenzhen University. In order to gather iGEM teams from various regions in South China for exchange and collaboration, and to break the knowledge lock brought by regions, we held the South China Conference in early May 2024, with more than 200 people attending the venue, reaching a historical high in scale. As the 8th South China Conference, this meeting established a stage for cooperation and exchange for teams in the South China region. During this conference, iGEM teams were able to establish meaningful connections with each other, thereby providing valuable and in-depth feedback on their projects. These feedback and suggestions will lay a solid foundation for the subsequent work of the teams, motivate team members to reflect on the shortcomings of their projects, and continuously modify and adjust their projects. We aim to continuously strengthen and maintain relationships between iGEM teams and cultivate a sense of community for team cooperation, hoping that this relationship will last long.

At the same time, our conference was held on the campus of Shenzhen University and was open to students on campus. This means that those with different professional knowledge backgrounds can participate in this conference and exchange learning with us. On the one hand, they can provide valuable suggestions from a public perspective, and we can also learn from them what the public really cares about, thereby continuously optimizing our project. On the other hand, this conference has made a great contribution to the establishment of a global iGEM community. Through the display of iGEM projects, more people can understand synthetic biology, participate in the iGEM competition, and use their own strength and wisdom to benefit more people through iGEM.

Learn more in Meetup .

Troubleshooting Cycle

E. coli Expression Verification

In the early stages of the experiment, we used E. coli to express the sweet protein Thaumatin and performed Western Blot (WB) to verify its correct expression. However, initially, we were not aware that E. coli might form inclusion bodies when expressing recombinant proteins, creating a kind of protein crystal within the bacterial cells, making it difficult to identify whether the target protein exists in the supernatant or the precipitate during the protein extraction process. Subsequently, we optimized the experimental plan and performed WB experiments on both the supernatant and the precipitate of the bacterial protein extraction solution. Compared with previous experiments, we obtained more accurate and reasonable results. This also provides valuable experience for subsequent iGEM teams working on bacterial strain expression of proteins.

Tomato Expression Verification

1. When conducting the tomato experiment, we used Agrobacterium infection to introduce our target gene into the tomato genome. Before the infection, we had to perform PCR on the bacterial colonies to ensure they contained the desired target gene. Initially, we had issues with excessive sample loading that caused sample stacking and failed to run the bands, and the Goldview nucleic acid dye we used was not suitable for long electrophoresis, resulting in an unclear agarose gel that could not display the bands. We then switched to Super Red nucleic acid dye, reduced sample loading, and obtained the expected bands.

2. After the Agrobacterium infection, we verified the expression of heterologous proteins in tomatoes (including Thaumatin and Brazzein). Brazzein is a small protein, so using conventional markers during loading and regular NC membrane during transfer did not yield the expected results. Therefore, after consulting literature, we switched to PDVF membrane and purchased new markers suitable for small proteins, and used a Tricine SDS-PAGE gel electrophoresis system that is more suitable for small proteins.

In addition, we continuously explored WB and summarized some experiences and lessons:

- During WB experiments, every step needs to be seamlessly connected to prevent the membrane from drying.

- Before developing, it is necessary to use TBST solution to thoroughly wash away the secondary antibody.

- After completing the WB experiment, the electrophoresis buffer does not need to be recycled.

- When blocking the membrane, it is necessary to block for two hours.

- Perform all operations on ice to prevent protein degradation.

Learn in more in Wet lab.