(1) Insight

Our project is too cutting-edge, and there are doubts about its feasibility.

(2) Initiate

Prof. Xue primarily focuses on biomass-based new energy production. We briefly introduced the project idea and some technical issues to him. Prof. Xue agreed with the technical approach of the project. He pointed out that the current development of synthetic biology has laid the foundation to support such innovations. He suggested that we set up redundant verification experiments at key nodes to enhance system stability and recommended referring to his team's relevant achievements in metabolic pathway regulation to optimize the matching between induction conditions and expression intensity. These suggestions provided strong support for the feasibility of the project.

(3) Improve

This interview has provided direction for the theme of our project. For subsequent topic selection, we will focus on: fully bio-based short-chain polyamides, so as to fill the market gap.

(1) Insight

Currently, the synthetic biology switches commonly used in metabolic engineering include light-controlled switches, temperature-inducible switches, toggle switches, and riboswitches. How should we choose?

(2) Initiate

After selecting the strain, we learned that in E. coli NT1003, lysine is the product of aerobic fermentation, while succinic acid only accumulates during anaerobic conditions. To achieve the simultaneous production of putrescine and succinic acid, we first need to design and select a dynamic regulation system that allows lysine accumulation during aerobic growth, with CadA (lysine decarboxylase) being inactive. Under anaerobic conditions, the system should then activate CadA to catalyze the conversion of lysine into putrescine. On one hand, from an industrial application perspective, he suggested that we find a suitable temperature-controlled switch, as temperature regulation in fermenters is relatively easier and more cost-effective. On the other hand, Prof. Liu also shared his team’s cutting-edge research on the artificial intrinsically disordered protein (A-IDP) encapsulation system and recommended using A-IDP encapsulated proteins.

*Feedback*

After a period of experimentation, we found that A-IDP failed to effectively encapsulate our CadA protein. We contacted Prof. Liu again via email, and he explained that this was likely because our protein significantly exceeded the size limit that A-IDP can encapsulate. He encouraged us to continue our efforts in synthesizing a larger variant of A-IDP. Unfortunately, we were ultimately unsuccessful in achieving this synthetic goal. Nevertheless, we still gained invaluable insights from the process.

(3) Improve

This interview has provided direction for the theme of our project. For subsequent topic selection, we will focus on: fully bio-based short-chain polyamides, so as to fill the market gap.

(1) Insight

Leaky expression persisted, and lysine accumulation still decreased as temperature increased.

(2) Initiate

Through prior exploration, we identified the more suitable promoter pJ23106, but several issues remained unresolved. We contacted Professor Zhang for assistance. Regarding our problem, Professor Zhang proposed using an RNA thermometer to regulate gene expression. He advised us to consult relevant literature and then attempt to incorporate it into our genetic circuit. We discovered that RNA thermometers are widely used in bacteria. Through our efforts, we successfully implemented regulation using an RNA thermometer, achieving excellent results.

(3) Improve

We promptly implemented this suggestion by introducing RNA thermometers (e.g., U7, U8) into our optimized thermal switch. The results were encouraging: the dual-switch system increased lysine accumulation to and enabled the efficient simultaneous synthesis of cadaverine and succinic acid, perfectly resolving the leaky expression issue.

(1) Insight

Through molecular docking, enhancing catalytic efficiency did not yield results. We felt somewhat confused and were unsure of how to proceed with the modification.

(2) Initiate

It is possible to focus on the properties of the CadA decamer, not modify the catalytic center, but instead indirectly improve enzyme activity by enhancing the stability of the decameric structure.

(3) Improve

After encountering obstacles in directly modifying the catalytic center (inactivation of multi-site mutants), we decisively shifted to Dr. Cai's strategy, focusing on stabilizing the decameric structure. Ultimately, we successfully obtained a high-performance mutant CadA(Y13C/P36C), which showed a significant improvement in enzyme activity compared to the wild-type.

(1) Insight

The model suffers from overfitting, and there is a need to construct an intracellular-extracellular coupling model to more accurately reflect the full-cell dynamics.

(2) Initiate

At the WCCE conference, we had the pleasure of meeting Prof. Mélaz Tayakout-Fayolle. She showed great interest in our project, particularly in our model. Following the conference, we continued our in-depth discussions via email.

*Feedback*

Later,we had an in-depth discussion with Prof. Mélaz Tayakout-Fayolle and demonstrated Python's curve_fit parameter fitting. The professor pointed out that relying solely on R² is insufficient and recommended incorporating regularization or cross-validation. Regarding the limitations of the Michaelis-Menten equation in the full-cell system, the professor suggested establishing a multi-module coupled ODEs model for "enzyme expression - intracellular reactions - transmembrane transport."

(3) Improve

After the discussion, we have clarified the optimization direction: adopting stricter algorithms to prevent overfitting, and building a multi-module coupled model. In the future, we will obtain key data through wet experiments for parameter fitting and validation.

(1) Insight

How to improve the prediction accuracy of the metabolic model for the putrescine/succinate co-production system? The current model shows discrepancies with experimental data and does not account for the regulatory effects of temperature-sensitive switches.

(2) Initiate

We presented the model framework and key targets to Dr. Feng. She pointed out that the value of static models lies in identifying trends rather than precisely predicting absolute values. Regarding the temperature-controlled switch, she suggested introducing regulatory constraints by converting known metabolic switches into flux constraints within the model, making it more aligned with the actual metabolic state of engineered strains.

(3) Improve

The optimization direction for the model was clarified: first, trust the model’s identification of key targets (such as gapA) to guide wet lab experiments for targeted modifications; second, explore the construction of an integrated rGEM model with regulatory logic. Future steps will involve using experimental data to constrain the model in a feedback loop, enhancing the model’s predictive and guiding capabilities through iterative dry and wet experiments.

(1) Insight

What is the future development potential of our project?

How can we improve its market competitiveness?

(2) Initiate

Yutong Zhang from Nest.bio responded in detail to our concerns about sponsorship. She noted that although the current starch-based route is mature, it is vulnerable to price fluctuations. In contrast, lignocellulose-based processes are more cost-effective and sustainable, potentially boosting the project's market competitiveness in the commercialization phase.She also suggested improving our investment appeal by presenting the project’s potential more clearly—through intuitive data visualization, exploration of commercial value, and innovative project positioning. She affirmed the commercial potential of our fully bio-based strategy.

(3) Improve

hrough this exchange, we gained a deeper understanding of how capital evaluates project value. Although due to time constraints we could not fully integrate this into the competition, we plan to continue exploring the market potential of PA54 and refine the business model in future iterations.

(1) Insight

How can synthetic biology research be aligned with industry regulations and applications?

(2) Initiate

To understand how academic results can transition into industry, we consulted Dean Zhansheng Wu from Xi’an Polytechnic University. He emphasized that bio-based materials—leveraging synthetic biology and biomass feedstocks—are a key strategy for achieving the "dual carbon" goals. While academic advances are progressing rapidly, transitioning to scalable industrial processes requires overcoming hurdles such as energy efficiency, cost, and equipment modification. This echoed points raised by experts from the China Textile Academy. We also realized that industrialization is not just a technical issue but must also align with national strategies for green development and meet safety and environmental regulations.

(3) Improve

This exchange made us more aware of the critical importance of safety and environmental sustainability. In response, we prioritized low-energy, low-pollution biosynthetic pathways in our lab experiments. Additionally, we incorporated the concept of “green production” into public outreach and academic communication to enhance the societal acceptance of bio-based products.