Wet Lab

The experimental work of this project makes outstanding contributions to the iGEM community by providing a series of validated standardized parts, reusable experimental protocols, and a scalable energy supply platform. This offers a significant foundation for subsequent teams to achieve efficient and stable energy metabolism in synthetic biology and industrial biotechnology applications.

1. Biological Parts

1. Standardized Biological Parts and Genetic Elements

Based on our novel ATP regeneration platform, V-CHARGEs (VLP-Coupled High-efficiency ATP Re-Generating Enzyme system), we have developed a variety of components for constructing V-CHARGEs. This provides the iGEM community and future researchers with a set of reliably performing genetic parts that have been thoroughly experimentally validated. These parts include the core encapsulation components, SlPPK-SP and CP-SpyTag fusion proteins, as well as SpyCatcher-Enzyme fusion proteins (e.g., FLuc, UCK, γ-GCS, GS) for external functional extension. The single and dual plasmid systems we constructed provide reusable and versatile genetic templates for in vivo assembly. The amino acid sequences for SlPPK, FLuc, UCK, γ-GCS, and GS serve as foundational elements for various reactions. The self-assembly mechanism of the Coat Protein (CP) and Scaffold Protein (SP), combined with the SpyTag/SpyCatcher covalent coupling strategy, provides an efficient and controllable technical pathway for protein encapsulation and multi-enzyme assembly.

2. Experiments and Protocols

2. Efficient Experimental System and Technical Protocols

Throughout the development of this project, we have progressively established and refined an efficient and reliable experimental system covering the entire workflow from protein expression and assembly purification to functional validation. This system combines practicality and reproducibility, providing systematic reference and methodological support for other iGEM teams engaged in similar research, specifically including the following three aspects:

(1) Novel Purification

(1) Protein Purification Innovation

We optimized the nickel-affinity chromatography process by introducing a dual-peristaltic pump system, effectively mitigating the issue of inclusion body formation common with large molecular weight proteins. This strategy significantly extended the interaction time between the target protein and the nickel resin, enhancing protein yield while reducing operational complexity, thus offering a new approach for the scalable production of soluble proteins.

(2) New Characterization

(2) Multi-dimensional Characterization and Refined Purification Strategies

Beyond conventional characterization techniques such as SDS-PAGE, Western Blot, DLS, TEM, and SEC, we innovatively employed low-concentration agarose gel native electrophoresis. This revealed differences in migration behavior between in vivo and in vitro assembly products, providing a new perspective for understanding assembly mechanisms. Regarding purification, by optimizing sucrose density gradient ultracentrifugation conditions, we successfully isolated structurally intact, high-purity V-CHARGEs particles. We utilized Dot Blotting for rapid assessment of assembly integrity, providing a reliable purification pathway for Virus-Like Particle (VLP) complexes.

(3) Systematic Validation

(3) Systematic Functional Validation System

Enzyme activity experiments confirmed that V-CHARGEs can continuously and efficiently regenerate ATP, demonstrating significantly superior stability and tolerance compared to free SlPPK under conditions of high concentraion of phosphate, protease treatment, and acidic stress. Furthermore, using various analytical methods including HPLC and ELISA, we validated the ability of V-CHARGEs to drive externally conjugated enzymes to catalytically synthesize products such as 5'-CMP and glutathione (GSH). This successfully achieved, for the first time, the functional coupling of "intracapsular ATP synthesis - extracapsular reaction driving", fully highlighting the platform's application potential in complex biosyntheses.

3. Universal Platform

3. Scalable Universal Platform for "Intracapsular Energy Generation - Extracapsular Synthesis"

We are the first to achieve the encapsulation of a tetrameric enzyme with a molecular weight as high as 200 kDa both in vitro and in vivo within the P22-VLP system, providing experimental evidence for expanding the application scope of this system. Additionally, we developed a novel and efficient in vivo assembly strategy for multi-enzyme complexes, providing other iGEM teams with a modular ATP generation platform for optimizing energy-consuming reactions. We demonstrated that encapsulating polyphosphate kinase via P22-VLP significantly enhances its tolerance and sustained activity in complex environments. The P22-VLP encapsulation effectively addresses industrial challenges such as phosphate inhibition and protease degradation. The established highly stable ATP regeneration system can substantially reduce production costs and is suited for long-duration, large-scale reactions due to its platform characteristics. These experiences are particularly suitable for teams focused on industrial translation for reference and adaptation.

In summary, the experimental work of the V-CHARGEs project not only contributes a rich diversity of new parts to the iGEM community but, more importantly, provides a high-quality, modular, universal platform based on the "Intracapsular Energy Generation - Extracapsular Synthesis" paradigm. This platform demonstrates significant advantages in enhancing enzyme stability, enabling continuous ATP supply, and promoting complex biocatalytic processes. Furthermore, the experimental experience and methodological system accumulated during the platform's construction provide a reliable foundation and valuable reference for subsequent iGEM teams working in synthetic biology, cell factory construction, and industrial biotechnology.

Dry Lab

The Dry Lab work of our project makes core contributions to the iGEM community by providing a research methodology spanning design, validation and application. This pioneers a novel paradigm for integrating computational biology with sustainable biomanufacturing and strategic industrial deployment.

1. Linker Designer System

The Linker Designer system represents a significant breakthrough and innovation over traditional linker property calculation software. While conventional programmes typically offer only basic physicochemical property calculations, this system establishes an intelligent, adaptive, multi-tiered prediction platform for the whole iGEM community.

Multidimensional Intelligent Screening System: Unlike traditional software with single-output computations, the Linker Designer system integrates multiple predictive algorithms and establishes a comprehensive scoring framework. By combining aggregation propensity prediction (APR identification, TANGO/WALTZ algorithms), secondary structure prediction (Chou-Fasman algorithm), and multi-model solubility integration (CamSol, Solubis weighted fusion), the system achieves intelligent screening and risk assessment of linker sequences. It automatically identifies high-risk regions, marks protective residue distributions, and provides users with actionable optimization recommendations.

Advanced Predictive Capability: The system goes beyond descriptive analysis to realize predictive modeling. Through the establishment of structure-function relationship models, it predicts linker performance in fusion proteins, including flexibility behavior, stability risks, and aggregation tendencies. This predictive capacity allows researchers to evaluate design feasibility prior to experiments, thereby greatly improving the success rate of fusion protein design.

Dynamic Database Self-Update Mechanism: Another key innovation lies in the system's self-learning and update capability. By integrating experimental datasets such as those from the Calgary iGEM 2023 LINKED project, the system can autonomously update its database according to user needs. This feedback mechanism enables continuous knowledge accumulation and enhances predictive accuracy, marking a true transition from a static computational tool to an intelligent platform.

2. Dual-model Strategy

2. Dual-model Exploration and Validation Strategy

We proposed and implemented a dual-model exploration and validation strategy, representing a significant complement and extension to traditional single-model simulation approaches. By combining discrete particle simulations with continuous ODE flux models, we established a closed-loop validation mechanism at both microscopic and macroscopic levels, while performing parameter inversion and order-of-magnitude verification. This ensures that key metrics, such as steady-state flux, the time constant of internal concentration, and final conversion, are highly consistent across both models. This strategy demonstrates methodological innovation: it preserves the underlying physical mechanisms and local details while maintaining engineering feasibility and effectively leveraging experimental data, thereby providing a reliable validation chain for dry lab investigations.

From an application perspective, the discrete model allows us to capture pore-level geometric bottlenecks and local dynamic behaviors, offering valuable guidance for microstructural optimization and mechanistic analysis. The continuous model complements this by enabling rapid parameter screening and steady-state assessment, which supports experimental design, prioritization, and efficient resource allocation. The synergy between the two models not only addresses the limitations inherent to each individual approach but also facilitates internal parameter interactions within the dry lab, enhancing model adaptability. This integrated approach allows the team to maintain scientific rigor while expanding the scope of dry lab validation tasks and improving the efficiency of parameter exchange between computational and experimental workflows.

Beyond the immediate project, our dual-model strategy carries broad illustrative significance. It provides a directly applicable methodological framework for problems in synthetic biology, including membrane transport, nanopore design, and metabolic network modeling. Moreover, it offers other research teams a cross-scale "microscopic-macroscopic-experimental" coupling approach, enhancing the reliability and generalizability of dry lab studies. Within the broader context of synthetic biology, this methodological contribution helps establish a standardized simulation-validation workflow, providing solid support for subsequent system design and experimental optimization.

3. Economic Benefit

3. Coupled Kinetics -Economic Benefit Model

We developed a Coupled Kinetics - Economic Benefit Model that integrates the experimental mechanism with enterprise-level economic decision systems, achieving a systematic mapping from laboratory parameters to industrial indicators. Based on the ATP-driven Glutathione (GSH) synthesis reaction, this model utilizes experimentally derived kinetic parameters to quantitatively evaluate the economic value of the V-CHARGEs technology, particularly in reducing energy consumption, alleviating substrate inhibition, and improving reaction yield. Through this framework, the performance advantages revealed by the Experimental Mechanism are translated into measurable Economic Benefit indicators, such as unit production cost reduction, annual profit growth potential, and investment payback period, thereby providing scientific and systematic decision support for the industrialization of the technology.

The contribution of this section lies not only in the economic modeling itself, but also in its methodological integration and innovative perspective. We move beyond the traditional dry-lab scope that focuses solely on reaction optimization or process simulation, and for the first time, couple the Experimental Mechanism with the Economic Benefit Framework to construct a data-driven bridge between scientific discovery and business application. The model directly maps technological improvements to their impacts on operational costs and profitability, helping enterprises identify the strategic value of the V-CHARGEs platform in energy-intensive biomanufacturing sectors. This modeling approach enables dry lab research outcomes to directly inform technological and market decisions, significantly enhancing their practical and strategic relevance.

Moreover, the model maintains a close resonance with the HP group's experimental work. Through P22-VLP encapsulation technology, the HP group effectively resolved the substrate inhibition challenge in ATP regeneration, providing critical experimental validation and parameter inputs for the Economic Benefit Model, thereby ensuring its robustness and reliability. Overall, this work establishes a framework for assessing the economic sustainability of green biomanufacturing, and offers a solid theoretical foundation for optimizing the industrial translation pathway of future synthetic biology achievements.

Human Practices

The contribution of 2025 BNU-China to Human Practices spans across the three major areas, Education, Entrepreneurship, and Sustainable Development, forming a unique model that integrates STEAM education, industrial consulting, and global collaboration.

1. Public Engagement

1. Bringing More People into the Embrace of Synthetic Biology

This year, BNU-China discovered significant shortcomings in STEAM education during our practical work: low participation of girls in education, lack of resources for rural children, and pronounced regional disparities in educational development. BNU-China focuses on the emerging field of synthetic biology, which is closely related to everyone's daily life, and attempts to bring more people into synthetic biology through widespread science popularization education. To systematically address the imbalance in STEAM education, we have collaborated with seven teams to create a regional STEAM education alliance system. This includes developing digital products like the "Teacher Training Platform"; creating customized courses for girls' education, designing targeted experimental projects and interactive activities; establishing the "ICD Cycle" mechanism (Inspire-Cultivate-Distribute); and founding the coordinating NGO UDGIC. We have developed storybooks, course outlines, teaching manuals, and more, with many of these classrooms reaching thousands of students through outreach teams.

Our project allows students of different age groups to have the opportunity to closely engage with and understand synthetic biology. Meanwhile, we have collected a wealth of real feedback from students, whose sincere and creative ideas not only inspire our project practices but also expand the direction for future course design and science promotion.

Our UDGIC platform's achievements have been internationally recognized, and we have been invited to share our experience on platforms such as the UNESCO Earth Charter Education Chair and the China-ASEAN Education Cooperation Week. We successfully promoted the use of the ESD Global Education Network to assist in the establishment of regional alliances, and officially launched the "iGEMers STEAM Alliance" initiative at the China-ASEAN Education Cooperation Week. Future iGEM teams can refer to our educational experience (see Education page) to extend science outreach further.

We hope to pass the STEAM education philosophy to future iGEM teams, helping them break through disciplinary boundaries and field constraints in science outreach, extending the reach of scientific communication to a broader societal space. At the same time, we will continue to focus on feedback and growth from rural teaching teams and benefiting students in iGEM 2025, summarizing real needs, accumulating practical experience, and enabling future teams to apply our experience more effectively in educational practices, achieving continuous optimization and intergenerational transmission of education.

2. Entrepreneurial Ideas

2. Entrepreneurial Reference Ideas

In the process of advancing the education alliance, we collected social feedback using the DBTL (Design-Build-Test-Learn) model, identifying two core value points. First, the ToB application potential of the V-CHARGEs technology. We found that its technological advantages in ATP regeneration and process optimization can provide production enterprises with cost optimization and ESG (Environmental, Social, and Governance) strategic consulting services, effectively achieving cost reduction and efficiency enhancement. Second, the deep integration need between education and industry. Education is not just about knowledge transmission; it also needs to align with industry demands, supplying enterprises with inclusive talents who have professional skills. Based on these insights, we innovatively constructed a "education + industry" dual-track development model. Using UDGIC as the foundation, we upgraded it into a comprehensive platform combining both education alliance coordination and ToB enterprise consulting functions. On one hand, it builds an education cooperation network for iGEMers in the China-ASEAN region; on the other hand, it provides ESG consulting and industry upgrading solutions for enterprises. The related practical steps are detailed in the Entrepreneurship page, covering market positioning, international cooperation, and technology commercialization, providing a full reference.