Contribution

Project Contribution Overview

The LEGO project makes multifaceted contributions to the iGEM community and the broader field of synthetic biology. Our work delivers a fully validated optogenetic toolkit for precise hormone control in mammalian cells, a functional software-hardware package that bridges clinical dosing with genetic circuit actuation, and a documented framework for translating stakeholder insights into concrete biological design.

Together, these contributions provide future teams with a comprehensive set of resources, from standardized parts and control systems to actionable methodologies, for developing the next generation of responsive, user-centric therapies.

A Characterized Modular Toolkit for Optogenetic Diabetes Therapy

1. Successful Implementation of a Complex, Multi-Component Optogenetic System

We have designed, built, and thoroughly validated a modular, reversible, and highly responsive optogenetic gene expression system centered around the iLID/sspB protein dimerization switch. This system integrates:

Core Optogenetic Switch: iLID and sspB as blue-light-inducible dimerization partners.

DNA-Targeting Module: The transcriptionally inactive DNA-binding domain of Gal4 (Gal4DBD).

Transcriptional Activation Module: VP64, a strong synthetic transcriptional activator.

Promoter System: A synthetic promoter with 5 tandem Upstream Activating Sequence (5xUAS) motifs for specific Gal4DBD binding.

We built and tested these components across multiple DBTL cycles, demonstrating not just initial function but also key properties like reversibility and nuclear localization.

This provides a fully characterized, modular "toolkit" for light-controlled gene expression, which has been robustly validated. Future iGEM teams or researchers can directly adopt this specific iLID/sspB-Gal4-UAS configuration for their projects requiring precise, reversible transcriptional control in mammalian cells, saving valuable time and effort required to construct such a system from scratch.

Parts Contribution

Basic Parts: We provide well-characterized coding sequences for iLID (BBa_25SVZLG1), SspB nano(BBa_25CKWQSO), Gal4DBD (BBa_25EWENZZ) Composite Parts: We constructed and tested functional fusion proteins, including NLS-miRFP670nano3-SspB (BBa_25T0DJLM), NLyn-miRFP670nano3-SspB (BBa_257KZSX5), NLuc-iLID-mCherry (BBa_25ZVZJXP), NLS-Nluc-iLID-mCherry (BBa_25FPKXWO) and NLS-Nluc-iLID-VP64-IRES-NLS-Gal4DBD-miRFP670nano3-SspB Light-inducible Transcription Activator (BBa_25XW3FSE)

Validation
We demonstrated:
1. Reversible dimerization within seconds of blue light exposure and rapid dissociation in the dark.
2. Stable nuclear localization of all components, even under prolonged illumination (18 hours), overcoming limitation of other optogenetic systems such as CRY2/CIB1.
3. Precise transcriptional control of reporter genes with low basal activity and high induction upon stimulation.

2. Pioneering the Use of Endogenous Bioluminescence for Optogenetic Control

We engineered a novel activation mechanism that replaces external blue light with self-generated, intracellular bioluminescence. This utilizes NanoLuc (Nluc) luciferase and its substrate fluorofurimazine (FFz). We demonstrated this mechanism by recruiting NIuc-iLID-mCherry to the plasma membrane through membrane-anchored sspB upon FFz addition.

This strategy offers non-invasive, deep-tissue control of synthetic biological systems, which eliminates the need for external light sources with limited tissue penetration. Teams working on mammalian therapeutics can adopt this NLuc/FFz-triggered system as a blueprint for creating autonomous or systemically controlled cellular devices.

Parts Contribution

Basic Part: We contributed a codon-optimized NanoLuc (Nluc) gene (BBa_25AWEP97).
New Composite Part: We built a NLS-Nluc-iLID-VP64 fusion (BBa_25XW3FSE) that integrates the light-generation capability into the light-inducible activator.

Validation
We demonstrated that:
Adding FFz to cells expressing iLID-sspB system triggers recruitment of iLID to membrane-anchored sspB (Fig. 12).
The response efficiency is comparable to external blue light activation.

3. Design and Implementation of Universally Secretable Human Insulin and GLP-1 in Mammalian Cells

Our team meticulously designed a human insulin variant capable of expression and secretion in generic mammalian cells, not just pancreatic β-cells. Through in silico simulations, we confirmed its structural consistency with native insulin and further validated its bioactivity in vitro. This engineered insulin was then integrated with a hydrolysis-resistant bioactive human GLP-1 to construct a functional dual-hormone expression cassette. This contribution directly addresses a key insight from our Human Practices research: the strong clinical demand and patient need for dual-therapy approaches that simultaneously manage both blood glucose and weight. By enabling the co-expression of both hormones from a single, implantable system, we provide a foundational framework for future teams to develop integrated therapies that move beyond single-target drugs and invasive injections, offering a path towards controllable, long-term, and patient-friendly management of metabolic diseases.

Parts Contribution

Basic Part: Engineered Human Preproinsulin (BBa_254SX1VP)
Design: We replaced the native, β-cell-specific PC1/3 and PC2 protease sites with a universal Furin cleavage site (RRKR), while retaining the native insulin signal peptide and all three native disulfide bonds.
Validation:
In silico analysis with Disulfide by Design 2.0 confirmed the preservation of all native disulfide bonds.
In vitro functional assays (PI3K/Akt pathway activation) proved the secreted insulin is fully bioactive.

Basic Part: Engineered Human GLP-1 (BBa_259V4OIU)
Design: We generated an active GLP-1(7-37) fragment with an A8G substitution for resistance to DPP-IV degradation and fused it to the Exendin-4 signal peptide for efficient secretion.

New Composite Part: Ins-IRES-GLP1-IRES-mCherry Dual-Hormone Expression Cassette (BBa_25EIL13E)
Design: A composite part combining both engineered hormones via an IRES element, followed by an mCherry fluorescent reporter, all under the control of the 5xUAS-Hsp70 inducible promoter (BBa_251FTTH1).

Validation
We demonstrated light-induced, coordinated co-expression of both hormones and the reporter at the mRNA (RT-qPCR) and protein (confocal imaging) levels.

Conclusion

We provide the foundational BioBricks to move beyond injectable therapies. Our parts enable the development of implantable, engineered cells that can secrete both bioactive insulin and GLP-1 in a light-controlled manner. This directly addresses the combination therapy paradigm that is the forefront of diabetes care. Future teams can use these characterized, modular parts to build advanced, multi-hormonal therapies for diabetes and other metabolic diseases.

A Practical Software-Hardware Package for Controlling Therapeutic Gene Circuits

Our Dry Lab contribution is a functional, open-source software and hardware package that translates a biological need—precisely controlling an optogenetic gene therapy—into a functional, user-friendly system. This integrated system is our solution to a common translational problem in synthetic biology: how do you make a genetic circuit that responds to a physical trigger (e.g. light) usable in a practical or clinical context?

1. A Ready-to-Use Tool for Dose & Actuator Control

We provide a fully developed web application (DiaPlan) paired with a programmable hardware controller (DiaLight). This combination is a practical contribution because it offers a complete workflow for therapies requiring controlled physical actuation.

What it does？

The DiaPlan software takes standard clinical inputs (weight, blood glucose, meal data) and performs two key translations:
It calculates personalized insulin/GLP-1 doses using established clinical formulas.
It converts these biological doses into specific commands (intensity, duration) for blue light illumination.

How it works？

PDF DiaPlan Software Instructions

The Hardware Link & Wet-Lab Integration: These commands are executed by the DiaLight, a wearable, ESP32-based device that we designed to be a cost-effective and programmable alternative to bulky lab equipment. Crucially, the DiaLight hardware is not just a conceptual design; it has been directly employed in our wet-lab experiments to provide controlled blue-light stimulation for cell cultures containing the LEGO genetic circuits. This direct application validates its functionality as a reliable actuator for optogenetic systems and bridges our computational models with biological validation.

Value for other iGEM teams: Any team working on an optogenetic system that needs in situ activation can use or adapt our hardware design and control software. The code is structured so that the core "dose-to-actuator" translation model can be recalibrated for different genetic circuits and their specific response curves.

2. Enhancing Understanding Through Interactive Simulations

To make our project more accessible, we developed a client-side simulator that replicates the control interface of our hardware. Visitors can input patient data, see the calculated therapy, and watch how it translates into a programmable light pattern. This interactive simulation helps demystify the core principle of our project, making it intuitive for users without synthetic biology expertise.

Interactive Simulation: We built a client-side simulator that replicates the full control interface of our hardware. This allows visitors to our wiki to experiment in real-time: they can input patient data, see the calculated therapy, and watch how it translates into a programmable light pattern. Interactive Hardware Simulation

Educational Value: This interactive experience does more than just demonstrate a feature; it teaches the fundamental operating principle of the entire LEGO system in an engaging way. It helps judges, other iGEMers, and the public grasp how a computational input leads to a biological output, bridging the gap between abstract concept and tangible application.

3. Built for Integration and Extension

Our software architecture is designed with modularity in mind, directly addressing criteria for the Best Software Tool prize.

Modular Design: The code is organized into separate, well-documented modules (e.g., Therapy Calculation Engine, Dose-to-Light Translator). This clear structure makes it easier for other teams to understand, modify, and extend the code for their own purposes. For example, the calculation engine could be adapted for different drug models.

API Compatibility: We demonstrated integration with an external Food Recognition API to improve carbohydrate counting accuracy. This serves as a working example for future teams on how to connect their tools to external data sources to enhance functionality.

Clinical Flexibility Feature: The Adaptive Rule Configurator is a direct contribution to usability and safety. It allows key variables (e.g. insulin sensitivity) to be adjusted, acknowledging that real-world medical protocols require professional oversight. This is a design consideration that other teams building clinically relevant software can learn from and implement.

4. Direct Contributions to the iGEM & Bioengineering Community

Our work provides concrete artifacts that future teams can use and build upon.

Open-Source Code & Hardware Specs: We release our complete software code and hardware design schematics under an open-source license. This allows others to replicate our system, troubleshoot based on our documentation, or use it as a starting point for their own control systems.

A Model for Closed-Loop Therapy Control: For the bioengineering community, we contribute a functional prototype of a user-in-the-loop control system. We show how to connect a patient-facing software interface to a programmable biological actuator. This end-to-end pipeline, from user input to physical stimulus, is a practical case study in translating a synthetic biology circuit into a potential therapeutic application.

Documented Workflow: Our detailed documentation, including the architectural diagram and the logic flow of our calculation engine, provides a clear blueprint for how to tackle the complex problem of interfacing clinical data with synthetic biological systems.

Conclusion

In summary, our contribution is a practical, open-source software and hardware package that makes the control of optogenetic therapies tangible. It serves as an educational tool to explain our project's core premise, a functional system that can be adapted by other teams, and a documented example of how to bridge the gap between computational dose calculation and physical actuation in a bioengineered system.

Integrated Human Practice Highlights and Legacy

Our Human Practices approach was integral to shaping the LEGO project. The main contribution we offer to future iGEM teams is not just the story of this process, but the concrete tools and documented decisions that resulted from it. Below, we summarize these practical resources that you can adapt, build upon, or use as a checklist for your own projects.

iHP overview

1. A Step-by-Step Framework for Connecting Surveys to Biological Design

Many teams conduct surveys, but the link from data to design can be vague. We provide a clear, actionable model for this.

What we did？

We created a "Stakeholder Insight to Design Feature" table. This table directly maps specific data points from our surveys (e.g., "Patient Q5, Q11: >40% feel troubled by injection routines") to a concrete design response (e.g., "Pursuing Non-Invasive Oral Activation") and the engineering rationale behind it.

Why it's a contribution？

This table is a reusable template. Future teams can use it to systematically justify their design choices to judges, providing a clear, auditable trail from identified need to a biological solution. It makes the integration of human practices explicit and verifiable.

2. Documented Pivots: How Expert Criticism Improved Our Project

We openly share how critical feedback from experts led to specific, significant project developments.

Clinical Feedback Driving Dry Lab Creation:

The Feedback: During our interview with Dr.Yu of Zhejiang Hospital, she challenged us on how we would achieve personalized dosing with a bioluminescence-based system, calling a one-size-fits-all approach "clinically irresponsible."
Our Pivot: This direct critique was the primary catalyst for creating our DiaPlan software and DiaLight hardware. Instead of abandoning our idea, we engineered a solution to the problem the expert identified.
Value for others: This is a case study for teams on how to treat expert criticism as a source of innovation, not a roadblock. It shows how a human practices activity can directly lead to the creation of a new and necessary dry lab component.

Peer Collaboration Leading to Wet Lab Optimization:

The Collaboration: In our meeting with the HiZJU-China iGEM collegiate team, they shared specific challenges they faced with the in vivo stability of GLP-1.
Our Pivot: Based on their shared experience, we introduced the Gly8 substitution into our own GLP-1(7-37) construct to enhance its resistance to degradation.
Value for others: This demonstrates a practical model for effective inter-team collaboration, where sharing specific technical hurdles leads to direct molecular-level improvements in both projects. It contributes concrete biological data (the use of Gly8) to the community.

3. Reusable Tools for Engagement and Implementation

We developed and refined several assets that other teams can utilize.

Dual-Audience Survey Structure: Our method of creating two parallel surveys—one for patients/the public and one for healthcare professionals—ensures that both user experience and clinical feasibility are captured from the start. Our wiki details the question flow and structure, which can be adapted for other health-related projects.
The "Adaptive Rule Configurator" in Software: A key feature added to our DiaPlan software after feedback from Dr. Xuelian Zhou from Children’s Hospital at Zhejiang University School of Medicine. This feature allows clinicians to view and adjust the key variables in our dosing algorithm. For future teams developing software for clinical settings, this is a crucial design consideration for ensuring clinical safety and adoption.
Four-Pillar Checklist (I.E.E.S.): We used a simple framework—Inclusivity, Education, Entrepreneurship, Sustainability—to periodically self-assess our project's broader impact. This serves as a practical checklist for future teams to ensure their work is responsible and real-world-ready.

4. Our Legacy of Concrete Artifacts

Our contribution is a set of tested resources: a design rationale table, a model for turning expert critique into innovation, a documented successful collaboration, and reusable tools for stakeholder engagement. We have shown that integrated human practices is about creating these tangible outputs that make a project stronger, safer, and more relevant. We encourage future teams to use and build upon these artifacts to make their own work more robust and responsible.