Our project has greatly benefited from the work of previous iGEM teams, whose contributions have shaped the foundations upon which we built. In this section, we aim to continue this collaborative spirit by highlighting the key reusable contributions generated by our team for future iGEM participants.
Labrys portucalensis F11 transformation
As part of our project, we initially aimed to work with the non-model bacterium Labrys portucalensis, known for its remarkable resistance to PFAS. However, to the best of our knowledge, no transformation protocol had been established for this species. Therefore, we needed to develop one in order to enable genetic manipulation of this strain. We were fortunate to benefit from the invaluable help of Véronique, who successfully achieved this feat.
A conjugation-based transformation protocol was developed to introduce plasmids from E. coli donor strains into Labrys portucalensis. Donor E. coli strains carrying various pSEVA plasmids (pSEVA621, pSEVA631, pSEVA651, pSEVA661) and the helper strain EGE214 were grown overnight in TSB medium supplemented with gentamicin (10 µg/mL) or kanamycin (50 µg/mL). Cultures of L. portucalensis were grown slowly for several days at 28 °C to reach an OD > 1. Equal optical densities of donor and recipient cultures were mixed, pelleted, resuspended in a small TSB volume, and spotted onto sterile 0.45 µm filters placed on TSA plates. After overnight incubation at 30 °C, filters were transferred to TSA plates containing ampicillin (100 µg/mL) and gentamicin (30 µg/mL) for selection of transconjugants. Colonies appearing after 5–7 days were reisolated on selective medium. Plasmid extraction from successful conjugations (notably with EGE214 + EGE460) confirmed the presence of recombinant plasmids by restriction digestion, demonstrating effective plasmid transfer into L. portucalensis through conjugation.
This represents a significant breakthrough that can be reused by all future teams working with this strain, whose remarkable characteristics continue to impress the scientific community studying PFAS.
One Health Matrix
What is the Ethical Matrix?
The Ethical Matrix (Figure 5) is a tool that maps all actors potentially affected by a synthetic biology project across three fundamental ethical principles:
- Autonomy: The capacity of stakeholders to make informed decisions and maintain control over factors affecting their lives
- Well-being: The physical, mental, and social health impacts on each stakeholder group
- Justice: Fair distribution of benefits and risks, addressing environmental justice and equity concerns
By evaluating each stakeholder group through these three lenses, teams can identify ethical tensions, prioritize concerns, and design projects that are not only scientifically sound but also socially responsible.
Why iGEM Teams Need This Tool
Synthetic biology projects inherently involve multiple stakeholders—from local communities and regulatory bodies to industrial partners and environmental ecosystems. Many teams focus primarily on technical feasibility, or overlook certain groups, like non-human actors. The Ethical Matrix addresses this gap.
How to Use the Ethical Matrix
Step 1: Identify All Stakeholders
Begin by listing every group affected by your project. Think broadly—include:
- Direct users (e.g. municipalities, industries, laboratories)
- Affected communities (e.g.residents near contamination sites, vulnerable populations)
- Regulatory bodies (e.g. environmental agencies, safety committees)
- Economic actors (e.g. competing technologies, suppliers, investors)
- Environmental actors (e.g.ecosystems, wildlife, plants)
- Scientific community (e.g.researchers, future iGEM teams)
- Ethical watchdogs (e.g. NGOs, advocacy groups, journalists)
Step 2: Analyze Each Stakeholder Against Three Principles
For each stakeholder, systematically evaluate:
Autonomy (+/−/+−)- Can they make informed decisions about the technology?
- Do they have agency in how it affects them?
- Are they consulted in development and deployment?
- Does the project improve or harm their health/safety/quality of life?
- Are there unintended consequences?
- What are the long-term impacts?
- Are benefits and risks fairly distributed?
- Does the project address or worsen existing inequalities?
- Who bears the costs? Who reaps the benefits?
Use (+) for positive alignment, (−) for negative, and (+−) for mixed or ambiguous impacts.
Step 3: Identify Conflicts and Prioritize
Look for patterns:
- Stakeholders consistently rated (−) across all principles require immediate attention
- Conflicts between stakeholder interests indicate areas needing mediation or redesign
- Groups with (+−) ratings need clearer communication or modified approaches
Step 4: Use Insights to Guide Project Decisions
Our experience with the Ethical Matrix led to concrete decisions:
- We chose not to partner with PFAS producers because doing so would compromise our commitment to environmental justice and community well-being
- We prioritized water treatment operators as partners since they aligned with our goals and could implement the technology responsibly
- We developed the One Health framework after realizing the interconnected nature of human, animal, and environmental stakeholder groups
- We created equitable pricing models after identifying justice concerns around municipal access to our technology
Figure 5 : Ethical matrix example.
