1. Overview

This year our project aims to advance DNA synthesis technology by developing a novel enzymatic approach utilizing Terminal deoxynucleotidyl transferase (TdT). Despite TdT's potential for template-free DNA synthesis, its random nucleotide addition limits its practical application. We will harness directed evolution to enhance the catalytic activity and capacity of the wild-type TdT from Zonotrichia albicollis (ZaTdT). Additionally, we will design and synthesize deoxynucleotide substrates with reversibly protected 3' termini, allowing for controlled nucleotide incorporation. This combination aims to achieve precise and orderly enzymatic DNA synthesis, addressing the efficiency and purity challenges associated with traditional chemical methods. By integrating these innovations, our project seeks to establish a greener, more efficient DNA synthesis platform, paving the way for advancements in genomics, synthetic biology, and data storage technologies.

2. Survey on Awareness of Synthetic Biology and DNA Synthesis Technology

We invite people to participate in the important survey aimed at assessing the public's understanding of synthetic biology and DNA synthesis technology. Synthetic biology is an interdisciplinary field that combines biology and engineering to design and construct new biological parts, devices, and systems. DNA synthesis technology, a crucial component of synthetic biology, enables the creation of artificial DNA sequences, which can have significant applications in medicine, agriculture, and environmental sustainability. We think this survey will help us gauge the level of awareness, perceptions, and knowledge regarding these cutting-edge technologies. The survey is anonymous and will take approximately 10 minutes to complete.

2.1. Questionnaire

Q1: What is your age range?

• ≤18
• 19-22
• 23-30
• 31-45
• 46-60
• ≥61

Q2: What is your highest level of education?

• Below high school
• High school/technical school
• Associate degree
• Bachelor's degree
• Graduate degree or above

Q3: Which country do you currently live in?

• Domestic
• Abroad (If you choose this option, please specify the country and skip question 3)

Q4: Which city do you currently live in?

• First-tier cities (Shanghai, Beijing, Guangzhou, Shenzhen)
• New first-tier cities (Chengdu, Chongqing, Hangzhou, Wuhan, Suzhou, Xi'an, Nanjing, Changsha, Tianjin, Zhengzhou, Dongguan, Qingdao, Kunming, Ningbo, Hefei)
• Second-tier cities and others

Q5: What is your major/professional field?

• Research and education
• Healthcare and health
• Biotechnology and pharmaceutical industry
• Information technology and communication
• Business and finance
• Law and policy-making
• Government and public administration
• Media and culture
• Non-profit organizations and social services
• Self-employed and freelance
• Other (please specify)

Q6: Is your major/profession related to biotechnology or related fields?

• Yes, related
• No, not related

Q7: How would you evaluate your understanding of biotechnology (especially DNA synthesis technology)?

• Have a deep understanding and mastery of professional knowledge
• Understand basic concepts and principles, and know some details
• Only have a basic understanding or have heard some terms
• Not very familiar, but willing to learn more
• Know nothing at all (skip questions 11-14)

Q8: Have you heard of the concept of "synthetic biology"?

• Yes
• No (skip questions 9-10)

Q9: Do you understand synthetic biology?

• Very well, have studied related professional knowledge or have relevant academic experience
• Have some understanding, roughly understand the basic definition
• Have heard of it, but unclear about the specific content

Q10: What are your main sources of understanding synthetic biology?

• News media
• Academic journals/books
• Professional courses
• Colleagues/friends
• Academic experience
• Other (please specify)

Q11: Which DNA synthesis method are you most familiar with?

• Phosphoramidite chemical synthesis method
• Enzymatic synthesis method
• Photochemical synthesis method
• Electrochemical synthesis method

Q12: What do you think is the biggest technical challenge facing current DNA synthesis technology?

• Limitations on synthesis length
• Control of error rates
• Improvement of synthesis efficiency
• Reduction of costs
• Other (please specify)

Q13: What factors do you think are the most important in the development of DNA synthesis technology? (multiple choices)

• Technology maturity and reliability
• Cost-effectiveness
• Innovation and cutting-edge nature
• Ethical and legal compliance
• Social acceptance and educational outreach
• Safety and environmental impact
• Other (please specify)

Q14: In which application areas do you think DNA synthesis technology has the most potential? (multiple choices)

• Biomedicine and disease treatment
• Agricultural biotechnology
• Industrial biomanufacturing
• Environmental monitoring and remediation
• Biological information storage
• Forensic science and judicial identification
• Food safety and testing
• Biological safety and defense
• Other (please specify)

2.2. Results

Based on the survey results, we can provide the following detailed analysis:

• Demographics of Respondents: The age distribution of respondents indicates a relatively young group, particularly with a higher proportion in the 19-30 age range. This age group is typically a critical period for academic and professional development, suggesting that they may have a heightened interest in biotechnology and synthetic biology. In terms of educational background, most respondents hold a bachelor's degree or higher, which provides a solid foundation for understanding complex concepts in biotechnology.
• Impact of Living Cities: Respondents are primarily concentrated in first-tier and new first-tier cities, which often have richer resources in the biotechnology industry and research. This may lead to more frequent exposure to biotechnology, thereby increasing their level of awareness.
• Relevance of Professional Fields: The survey shows that many respondents work in fields related to biotechnology, particularly in research and education, healthcare, and the biotechnology and pharmaceutical industry. This professional background enables them to provide more specialized and in-depth insights when answering questions about DNA synthesis technology and synthetic biology.
• Understanding of Biotechnology: Most respondents indicate that they have a certain level of understanding of biotechnology, especially DNA synthesis technology, with a significant portion possessing professional knowledge. This suggests a strong interest and willingness to learn more about the field, which may facilitate their further exploration in future career development.
• Awareness of Synthetic Biology: The majority of respondents have heard of synthetic biology and have a certain level of understanding of it. This indicates that synthetic biology, as an emerging field, is gradually being recognized and accepted by more people, particularly among professionals related to biotechnology.
• Familiarity with DNA Synthesis Methods: Respondents show varying degrees of familiarity with different DNA synthesis methods, indicating differences in their knowledge reserves regarding technical details. Understanding the advantages and disadvantages of various synthesis methods is crucial for advancing technology and its applications.
• Technical Challenges and Development Factors: Regarding the challenges facing DNA synthesis technology, respondents generally believe that limitations on synthesis length and control of error rates are the most significant issues currently. At the same time, they also emphasize the importance of technology maturity, cost-effectiveness, and ethical compliance as factors in technological development. This suggests that respondents view technological advancement not only from a technical perspective but also consider its social and ethical implications.
• Perceived Application Potential: Respondents believe that DNA synthesis technology has broad application prospects in fields such as biomedicine, agricultural biotechnology, and environmental monitoring. This reflects an optimistic attitude towards the potential of biotechnology to address real-world issues, particularly in tackling global challenges such as diseases, food security, and environmental protection.

Overall, the survey results reflect a high level of interest and awareness of biotechnology and synthetic biology among respondents, especially in groups with higher professional backgrounds and educational levels. This provides a solid foundation for future research and applications in related fields while also highlighting key issues that need to be addressed in current technological development.

3. Deep consulting with a university professor about our project and experimental design

When we proposed our project design and specific plan, we consulted a Chemical biology Professor Hao Sun from Nanjing Agricultural University about the feasibility of our experimental design and sought optimization opinions and suggestions. Based on multiple discussions with Professor Sun, we have gained a clearer understanding of our planned design and potential future application directions. We have summarized some specific and feasible optimization solutions.

3.1. Feasibility Analysis and Suggestions for Optimization

3.1.1. Directed Evolution of TdT

Feasibility Analysis:

• Source Selection: Starting with ZaTdT is a sound choice given its superior catalytic activity compared to other TdT sources. This should provide a strong foundation for generating effective mutants.
• Molecular Docking: Utilizing molecular docking to predict mutations that may enhance activity is a rational approach. Ensure that the docking software used is well-validated and that the scoring functions accurately reflect the binding affinities and catalytic efficiencies.
• Site-Directed Mutagenesis: This technique is well-established and should yield a variety of mutants. Consider using a library approach to create a diverse set of mutants that can be screened for improved activity.

Suggestions for Optimization

• High-Throughput Screening: Implement high-throughput screening methods to quickly evaluate the catalytic activities of the ZaTdT mutants. This can significantly accelerate the identification of promising candidates.
• Characterization of Mutants: In addition to catalytic activity, assess other characteristics such as thermal stability, substrate specificity, and reaction kinetics to identify mutants that may have advantageous properties beyond just activity.
• In Vivo Expression: While E. coli BL21(ED3) is a good choice for protein expression, consider exploring other expression systems (e.g., yeast or insect cells) that might provide better folding or post-translational modifications for the TdT enzyme.

3.1.2. Design and Synthesis of Deoxynucleotide Substrates

Feasibility Analysis:

• Chemical Modification: The approach of adding a light-sensitive protecting group to the 3' ends of deoxynucleotides is innovative and aligns well with controlled DNA synthesis strategies. The specificity of the protecting group will be crucial for the success of this method.
• Testing with TdT: Validating the ability of wild-type and mutant TdT to add modified substrates to single-stranded DNA is a logical next step. This will provide insights into the functionality of the modified nucleotides.

Suggestions for Optimization:

• Characterization of Protecting Groups: Carefully select and characterize the protecting groups to ensure they can be efficiently removed under the desired conditions without affecting the integrity of the nucleotide or the DNA chain.
• Optimization of Reaction Conditions: Experiment with various reaction conditions (e.g., temperature, pH, ionic strength) to optimize the activity of TdT with the modified substrates. This may help in achieving higher incorporation rates.
• Control Experiments: Include control experiments with unmodified deoxynucleotides to benchmark the performance of the modified substrates and to rule out any non-specific effects.
• Light Exposure Parameters: Establish the optimal light exposure conditions (wavelength, intensity, duration) for deprotection to ensure that the reaction proceeds efficiently without damaging the DNA or affecting TdT activity.

3.2. Conclusion

The proposed experimental design is promising and aligns with current trends in chemical and synthetic biology. By implementing the suggested optimizations, the project can enhance the likelihood of success in achieving the desired outcomes of improved TdT activity and controlled DNA synthesis. Regularly reviewing progress and adapting strategies based on preliminary results will also be crucial in navigating any challenges that arise during the experiments.

4. Interview

Based on our project design and preliminary research results, we seek to engage with experts from different fields to discuss new DNA biosynthesis technologies and their potential impacts and driving effects on synthetic biology and other related areas.

4.1. Interview with Xi'an Mark Medical Technology Co., Ltd.

In this interview, Mr. Ma emphasized the application of 3D printing technology in the medical field and its potential for integration with DNA synthesis technology. By combining these two technologies, we can promote the development of personalized medicine, improve treatment efficiency, and foster innovation in biomaterials, thereby enhancing the treatment experience for patients. The summary and significance of this human practice activity are as follows:

1. Promoting Interdisciplinary Collaboration: This activity showcased the close connection between biotechnology, medicine, and engineering. Through the exchange and collaboration among experts from different fields, new ideas and solutions can be stimulated, advancing the development of synthetic biology.

2. Facilitating Diverse Technological Applications: The integration of DNA synthesis technology and 3D printing can lead to innovative applications across various fields, including medicine, agriculture, and the environment. For example, the development of personalized vaccines and the manufacturing of tissue engineering scaffolds will enhance the efficiency and effectiveness of biotechnological applications.

3. Enhancing Public Awareness and Acceptance: Through human practice activities, the public's understanding of synthetic biology and DNA synthesis technology will be improved. This increased awareness can help dispel misconceptions and fears surrounding new technologies, promoting societal acceptance of biotechnology.

4. Advancing Education and Training: Educational tools that combine DNA synthesis technology with 3D printing can provide students with a more intuitive learning experience, igniting their interest in biological sciences. This will cultivate future scientists and engineers, further advancing the field of synthetic biology.

Addressing Future Challenges: As biotechnology rapidly evolves, the ethical, legal, and social issues it faces are becoming increasingly complex. This activity emphasizes the importance of considering these issues when promoting technological applications to ensure safety and sustainable development.

In summary, this human practice activity not only enhanced our team's understanding and application of the potential new technologies generated by this project but also laid the foundation for the future development of DNA synthesis technology and synthetic biology. Through interdisciplinary collaboration and public participation, we can better address future challenges and achieve sustainable development in biotechnology.

4.2. Interview with Dr. Hocquette

The interview first discusses the current status of cultured meat technology and the challenges it faces. Although cultured meat has potential in terms of nutritional value, environmental impact, and affordability, the technology is still immature. Researchers need to consider multiple factors when mass-producing cultured meat, including public acceptance, the impact on agriculture and the meat market, and how to replicate conventional meat. The interview mentions that consumer trust in cultured meat needs to be built through transparent research results. However, private companies face difficulties in communicating research findings transparently because they want to protect their intellectual property. Additionally, many people are concerned that mass production of cultured meat could negatively impact the conventional meat market.

In terms of environmental impact, while cultured meat is believed to have potential benefits, its carbon footprint is difficult to track, and the production process can release a significant amount of carbon dioxide. The document points out that scientists can only estimate its carbon emissions, and current estimates may be lower than the actual situation.Regarding the nutritional value of cultured meat, it is noted that it may lack some micronutrients found in traditional meat, and there is no data on the digestibility of these nutrients. Furthermore, the texture and aging process of cultured meat have not been adequately studied.

In summary, this emphasizes the challenges of cultured meat technology, including cost, public acceptance, nutritional value, and environmental impact, and highlights the need for more research and transparency to advance this emerging technology.

We also discussed how the integration of new DNA synthesis technology with cultured meat technology could significantly advance the development of synthetic biology. Here are several potential points of integration:

1. Optimizing Nutritional Components: New DNA synthesis technology can be used to design and synthesize specific genes to enhance the nutritional value of cultured meat. For example, gene editing techniques could increase the content of micronutrients such as iron and vitamin B12 in cultured meat, making it closer to the nutritional profile of traditional meat.

2. Improving Production Efficiency: By improving gene expression during the cell culture process, the proliferation rate and growth efficiency of cells can be enhanced, thereby reducing the production costs of cultured meat. New DNA synthesis technology can help develop more efficient cell lines and culture conditions.

3. Enhancing Texture and Flavor: Utilizing DNA synthesis technology, genes that influence muscle cell differentiation and maturation can be designed, which may help improve the texture and flavor of cultured meat, making it more similar to traditional meat.

4. Environmental Impact Assessment: New DNA synthesis technology can assist scientists in establishing more precise models to evaluate the carbon footprint and environmental impact of cultured meat. By optimizing various stages of the production process at the genetic level, the overall environmental impact may be reduced.

5. Public Acceptance: Through transparent research and technological demonstrations, the production process of cultured meat combined with DNA synthesis technology can more easily communicate its safety and advantages to the public, helping to dispel misconceptions and resistance towards cultured meat.

In summary, combining new DNA synthesis technology with cultured meat technology not only addresses some of the current challenges faced by cultured meat but also has the potential to further advance synthetic biology and promote sustainable food production.