Contribution ​

Introduction ​

iGEM is a community built on collaboration and warmth, where every step forward is supported by a tradition of sharing and mutual growth. As we began our project this year, we were met with the generous guidance of those who walked this path before us and the spirited fellowship of our peers. Their wisdom lit our way, and their steadfast support fostered an environment where innovation could thrive.

Staying true to this spirit, we are committed to giving back by sharing not only our findings but also the lessons learned from the obstacles we faced. It is our sincere hope that these contributions will serve as a stepping stone for the teams that follow, helping to sustain a lasting culture of cooperation. Together, we can expand the frontiers of synthetic biology and open new horizons for the scientists of tomorrow.

Wet Lab ​

In our project, we explored SAM concentration-responsive aptazymes with the aim of utilizing them to sense SAM levels, thereby achieving negative feedback regulation of SAM in cirrhotic cells and maintaining it consistently within a normal physiological concentration range. Based on existing literature, we fused the SAM VI riboswitch with the HDV ribozyme to create the Aptazyme SAM VI. This construct was subsequently modified and evolved through adjustments in the number of neck base pairs and variations in base composition. After multiple rounds of simulation and experimental validation, we successfully developed a concentration-responsive aptazyme, which was applied and tested in our established cirrhotic cell model.

Parts ​

We have registered three engineered aptazymes with SAM concentration-responsive behavior in the iGEM Registry of Standard Biological Parts: BBa_25VD7R1M, BBa_25WN8EQU, and BBa_2535VKHV. The ribozyme activity can be allosterically regulated by the aptamer ligand. SAM binding to the aptamer triggers self-cleavage of the ribozyme component. Integration of this cassette into the 3'UTR enables selective gene regulation in mammalian cells.

Since the upstream CDS region of this component can be replaced with any gene of interest, it can be utilized for targeted gene expression regulation in cirrhotic cell models to achieve diverse biological functions. This system provides a cost-effective molecular switch for modulating metabolic pathways in hepatic cells, while offering broad application potential for future research on cirrhosis.

Each registered part is accompanied by comprehensive characterization data, documenting the functional properties of the SAM-responsive aptazyme components we have developed. This detailed profile offers future teams a clear and systematic understanding of our findings, enabling them to quickly grasp the operational nuances of our designs and explore how these elements may be adapted or extended within their own projects.

Experimental Protocols ​

Our project utilized various SAM riboswitches and ribozyme components. The modification and evolution of aptazymes represent a highly complex endeavor, and our engineering approach can provide future teams working with aptazymes with additional modification strategies and methodologies. Our experimental design incorporates multiple experimental procedures for characterizing aptazyme function. Following our experimental protocols can help other researchers save significant time and costs in experimental design.

This collection of methodologies represents more than a simple compilation of experimental steps—it embodies our iterative process of optimization, capturing the invaluable insights gained from each setback. We have structured this resource to be intuitive and actionable for subsequent research teams, featuring comprehensive procedural guides, essential safety notes, and practical troubleshooting advice that proved instrumental to our successful implementation.

These materials have been integrated into the engineering database of our shared platform, where they remain readily available to researchers interested in building upon our work or pioneering new directions in the field of aptazyme-based metabolic regulation.

Dry Lab ​

Our work contributes to the iGEM community primarily via three pillars, supplemented by broader open-source support and reproducibility.

Core Contributions ​

1. Software – RNA-Factory: an original, integrated, user-friendly RNA analysis platform ​

To lower the barrier for teams to access modeling tools, we developed RNA-Factory, which consolidates multiple functionalities with clear documentation:

• Provides a unified interface for structure prediction, interaction prediction, sequence/structure design, and result visualization.
• Employs LLM + RAG and agent mechanisms to assist model selection, explanation, and result interpretation.
• Integrates DeepRPI, MPNN, and other modules behind the scenes, so users only need to input simple files/sequences, not manage multiple models manually.

2. Molecular Dynamics Simulations — Hand-on Manual + Robust Pipeline ​

We developed a multi-stage, high-throughput molecular dynamics (MD) simulation pipeline with comprehensive documentation, enabling easy reproduction and adaptation by other research teams.

• Structure preparation and modeling: RNA structures were generated using multiple computational techniques, followed by structure filtering to ensure model accuracy and stability.
• Simulation setup: Standardized MD protocols were employed with appropriate force fields and physiological conditions to closely mimic experimental environments.
• Post-simulation analysis: We conducted MM/PBSA free energy calculations, principal component analysis (PCA), free energy landscape (FEL) mapping, and related analyses to identify key residues and characterize conformational dynamics.
• Screening and experimental validation: By ranking riboswitch candidates based on simulation-derived metrics, we guided downstream experiments. Computational predictions correlated well with experimental outcomes, validating the robustness of the workflow.
• Documentation and reproducibility: A complete set of annotated scripts, parameter files, and example datasets are provided in the user manual, allowing researchers without extensive computational experience to reproduce the pipeline with ease.

3. DeepRPI — Transparent Predictive Model with User Guidance ​

We provide a RNA–protein interaction predictor (DeepRPI) with complete documentation:

• Clear model architecture & input encoding.
• As a functional screening tool: users can input RNA and protein sequences and get binding/non-binding probability scores. This allows preselecting promising pairs before expensive experiments.
• Benchmarking & performance disclosure: we publish training/validation curves, ROC etc., so users can see baseline performance and compare improvements.
• Integration in the software platform: DeepRPI is fully wrapped into our RNA-Factory platform, so users can call it via a user interface without handling model internals or dependencies.

Secondary / Supporting Contributions ​

• Open Source & Reproducibility All code, models (DeepRPI, MPNN, etc.), simulation scripts, ODE / kinetics pipelines, and platform code are openly released with clear licenses.
• Comprehensive Reproduction Guides Each module (simulation, prediction, software) comes with step-by-step instructions, annotated code, parameter files, and example datasets, enabling future teams to reproduce results without reinventing the wheel.
• Lowered Learning & Debugging Cost Through manuals + ready wrappers + integrated platform, we significantly reduce the barrier for teams lacking deep computational background to adopt these tools in their projects.
• Bridging Experiment & Computation Our modeling and software tools are not just afterthoughts — they actively feed into experiment design. Subsequent teams can continue using our loop, accelerating cycles and improving efficiency.

Inclusivity Guide ​

This inclusivity guide compiles our experiences and reflections from Human Practice work, exploring from multiple perspectives how to truly implement "inclusivity". The content covers inclusive design of language and content, accessibility and barrier-free dissemination of information, respect for cultural and linguistic diversity, strategies for event organization and public participation, construction and improvement of communication channels, as well as how to balance cost and impact when resources are limited. Each part is based on actual exploration and trial and error, not only documenting the path to success but also honestly presenting the difficulties and trade-offs we have encountered.

Our guide is available in multiple languages and comes with a short explanatory video. We hope these efforts can help future igem teams get started quickly.

We hope this guide can offer some practical assistance to future iGEM teams when conducting Human Practice - whether it is planning the project framework, considering the needs of the audience, or reflecting on their own positioning, they can all find references and inspirations from it. If it can make you stop to think and re-examine the meaning of "inclusiveness" at some point, even if it's just a momentary inspiration or a minor adjustment in direction, it has already fulfilled our original intention.