Project Phases

"Concern for man himself and his fate must always constitute the chief objective of all technological endeavors... never forget this in the midst of your diagrams and equations." - Albert Einstein

Tommaso Varaschin

Padua iGEM 2023

The Bologna iGEM team 2025 was inspired by Tommaso Varaschin, a member of the Padua iGEM 2023 team. Their project, P.A.S.T.A. (Phage Assisted Silencing Tool Against AMR), employed bacteriophages to deliver CRISPR interference (CRISPRi) modules to silence antibiotic resistance genes, thereby decreasing selective pressure on microbial communities and preserving the host microbiota (see project wiki). After being introduced to the competition framework, the Bologna team began developing its own project concept.

Mirko Zaffagnini

Professor, University of Bologna

To ensure feasibility within the iGEM timeframe and to maximize scientific relevance, the team sought advice from faculty members of the University of Bologna. Through these consultations, the team established a collaboration with Prof. Martina Cappelletti, whose research focuses on Rhodococcus species. Together, they identified a project aimed at leveraging Rhodococcus's metabolic versatility. Additional discussions with Prof. Mirko Zaffagnini, an expert in metabolic engineering, guided the team toward the idea of reconstructing and modifying Rhodococcus metabolic pathways to degrade a target environmental pollutant.

Marco Candela

Professor, University of Bologna

Federico Manuel Giorgi

Professor, University of Bologna

To support this goal, the team also considered the development of a computational tool for metabolic pathway prediction. Together with Prof. Marco Candela and Prof. Federico Manuel Giorgi we agreed the tool should integrate information from biological databases and contextualize it within the metabolic framework of Rhodococcus, providing a basis for rational pathway engineering.

Finally, we asked ourselves whether engineering Rhodococcus could negatively affect its triacylglycerol production. To make sure this was not the case, we needed to find a way to quantify the bacterium's products, so we turned to the Department of Chemistry in Bologna to find chemists willing to support our cause.

The Bologna iGEM team 2025 was born from the students' ambition to create something new and meaningful. Aware of the limitations of our expertise as a student-led team, we actively sought guidance from the broader scientific and non-scientific community around us. We engaged directly with individual experts across various fields to gain informed perspectives on the challenges and implications of our project. Our approach was shaped by a simple yet effective model: formulate a clear question based on our uncertainties, carefully listen to the answers provided by experts, and design concrete actions in response. This iterative dialogue with the community not only strengthened our project but also fostered a culture of responsibility, openness, and collaborative problem-solving.

Figure 1. Our three-step model that enabled us to have an efficient dialogue with the experts. The first step is to accurately pinpoint our doubts and make sure they are expressed as clearly as possible. Then, we write down the answers, and only afterwards do we look back to reflect and evaluate their words. The outcome of this iterative process must always be a concrete action, whether it is deciding to continue on our initial path or trying a new solution.

"See a need, fill a need." Bigweld – "Robots" (2005), 20th Century Fox

We consulted with several experts in order to understand how to make our idea real: we wanted to develop a chassis capable of simplifying the work of researchers by providing a reliable and versatile platform. We also wanted this type of chassis to be able to generate a meaningful and positive impact on the community and the environment.

During the design phase, we made sure to consider biosafety aspects by consulting an infectious disease doctor in order to verify both the safety and the functionality of our chassis. At this stage, we also reflected on how to implement our HERO in order to maximize its positive impact. We applied the same principles to the design of the CAPE tool. At the very beginning of our research we found a gap in the synthetic biology toolkit: comprehensive open-source pipelines that integrate both predicted and annotated pathways were lacking. From the beginning, we had some key principles in mind: we wanted to create a platform accessible to everyone, precise, biologically accurate, and still able to automate the researcher's work.

Infectious Disease Doctor - Dr. Giovanni Dolci

Assessing Biosafety

❓ We consulted him in order to assess the biosafety of engineering Rhodococcus opacus PD630, by evaluating its potential pathogenicity in humans, its antibiotic resistance profile, and the likelihood of horizontal gene transfer.

🔧 We integrated the antibiotic resistance gene into the genome of R. opacus PD630 to minimize the risk of horizontal gene transfer. This decision reflects our commitment to safe and responsible bioengineering, especially in view of potential future applications of HERO.

That's the trouble with science. It's never done. Always upending itself. Ruining perfect systems for the little inconvenience of them being wrong. Brandon Sanderson, "Rhythm of War" (2020)

The design phase of our project led to the development of three distinct yet interconnected components:

HERO: A chassis engineered for bioremediation
CAPE: A predictive tool for the metabolic pathway analysis of Rhodococcus
HELMET: A biosensor designed to quantify specific TAGs

Throughout this process, interdisciplinary dialogue with professors and experts in biotechnology, computer science and chemistry fostered a synergy that we consider essential to the successful development of our project.

Although each component follows its own research trajectory, they are all deeply interlinked in both purpose and functionality.

Let's take a closer look at how each of them evolved throughout our journey!

HERO

Our biotechnology chassis for bioremediation.

Hector M. Alvarez

Validating Our Engineering Strategy

❓ We sought Professor Alvarez's expert opinion to validate our conceptual approach and experimental design for engineering Rhodococcus opacus.

🔧 His positive feedback gave us confidence in our direction. We proceeded with the design of the genetic construct.

Umberto Genchi

Troubleshooting and Expanding Genetic Tools

❓ We sought Umberto's advice to overcome technical challenges in amplifying our GC-rich genetic construct.

🔧 Thanks to his input, we improved amplification by switching to ExTaq polymerase.

Hector M. Alvarez

Promoter Testing

❓ We asked Prof. Alvarez for his opinion on our strategy to improve gene expression in Rhodococcus opacus.

🔧 We decided to test two classes of promoters: PLac and P2.

CAPE

Our computational tool for metabolic pathway prediction.

Thomas Duigou

Computational Pathway Design

❓ We consulted him to validate our computational approach for metabolic pathway design.

🔧 We redesigned our tool to balance automation with user decision-making.

HELMET

Our biosensor for TAG quantification.

Roberto Aureli - Alchemy Srl

Quantification Strategy

❓ We asked Alchemy for support in quantifying Rhodococcus opacus PD630 byproducts.

🔧 We decided to focus on TAG detection using a biosensor approach.

"The scientific man does not aim at an immediate result. He does not expect that his advanced ideas will be readily taken up. His work is like that of the planter: for the future." Nikola Tesla

HERO IMPLEMENTATION in wastewater treatments

We dedicated a significant portion of our time to identifying the most effective application for HERO. Driven by the desire to bridge the lab-to-market gap, we engaged with various stakeholders in the water treatment sector. Our goal was to evaluate the feasibility of integrating Rhodococcus into a functional system and to determine what modifications would be required.

As our research progressed, we became particularly interested in membrane bioreactor (MBR) systems as a promising platform for our chassis. To deepen our understanding, we consulted with industry professionals, including operators of biological treatment plants such as Acque del Chiampo and ENEA, as well as membrane manufacturer Everblue Srl. These conversations focused on uncovering the key challenges currently faced in wastewater management, with the aim of developing solutions that are both impactful for communities and aligned with real-world demands.

Acque del Chiampo

Public Utility Company - Arzignano, Italy

❓ Are HERO's growth requirements compatible with treatment plant conditions? Could HERO target PFAS and chromium from tannery effluents?

ENEA

National Agency for Energy & Sustainable Development

❓ What are setup and operational costs? How to produce large inoculum quantities? Are there GMO case studies in full-scale facilities?

Everblue

Advanced Water Filtration Systems

❓ How does HERO perform in MBR systems? Impact on membrane fouling? Interactions with indigenous microbial consortia?

To better understand how to implement our chassis, we sought to address some of these questions by consulting with an expert who possessed greater knowledge than ourselves.

Eva Donini

PhD Candidate - Environmental Microbiology

University of Bologna. Research focuses on Rhodococcus bioremediation, microbial consortia for filter regeneration, and biochar carriers for soil bioremediation.

Possible Actions

Although we have not yet been able to turn these ideas into concrete applications, we now have a clear understanding of the key aspects to consider as we face the next stages of development. Thanks to the insights gained from Acque del Chiampo, ENEA, Everblue, and Dr. Eva Donini, we identified several strategic directions for HERO's potential implementation:

Post-treatment integration in existing plants to target persistent pollutants like PFAS and heavy metals, minimizing interference with primary biological processes.
Application in MBR systems, optimizing strain behavior to reduce fouling and improve compatibility with membrane filtration technologies.
Use of pre-adaptation bioreactors to boost degradation efficiency by gradually exposing HERO to target contaminants.
Economic and technical feasibility studies, focusing on scalability, inoculum production, and cost-efficiency, particularly in industrial settings.

These actions represent promising next steps in aligning HERO with real-world wastewater treatment challenges.

HERO's impact on society

Silvia Zullo

Associate Professor - Philosophy of Law and Bioethics

University of Bologna. We consulted her to understand whether our project could have a negative impact on the environment or society.

Focus areas: Dual use and risk assessment - considering whether technology designed for beneficial purposes could be misused to cause harm.

🔧 We reflected on how our technologies could be misused and implemented appropriate safeguards.

Bioremediation Survey

We decided to ask these questions directly to the public by creating a short survey about our project and bioremediation, which is the application we are most interested in pursuing in the future. The survey collected 211 responses.

Key findings:

Almost two thirds of participants had no idea what bioremediation is
Participants believe that the use of bacteria in industry will become increasingly common
They agree it is important to invest in this field and use bacteria in enclosed facilities such as wastewater treatment plants
Most striking result: One third of participants said that calling the bacterium "genetically modified" instead of "engineered" would make them change their opinion. This indicates that word choice is important, but even more important is filling the gap of knowledge, since the two terms are practically the same.

Our action: This finding, combined with the fact that participants do not know about bioremediation yet want to learn more but have not had the opportunity to read about it, led us to focus on the educational aspect of our project, particularly with high school students at Liceo Scientifico Augusto Righi in Cesena.

HERO on the market

Business Development

We attended several webinars on entrepreneurship and intellectual property offered by the Knowledge Transfer Office at the University of Bologna. Thanks to these sessions, we gained skills that enabled us to conduct a market analysis and develop a business plan, which we presented at the Start-Up Day, where we won a prize for our project presentation.

Figure. Business plan of the HERO project presented at the Start-Up Day.

Current status: After developing the plan, we all agreed that our project was still in the early stages of development, so it was too risky to focus too heavily on this route. However, if we continue to grow in the future, we will be able to navigate the market with more confidence, having already defined our goals and established a foundation in entrepreneurship.

CAPE Strategic Direction

During this phase, we also reflected on the strategic direction of CAPE, questioning whether it should evolve toward commercialization or remain a fully open-source research tool. Given the promising applications of CAPE in synthetic biology and bioremediation, we sought further guidance from Dr. Thomas Duigou to evaluate the balance between open science, intellectual property protection, and entrepreneurial potential.

Which communities would be most interested in our project, and are there any potential negative impacts we should consider? Should we patent our tool, and could CAPE evolve into a start-up?

The synthetic biology community would be the primary audience for our project. However, the ability of CAPE to support the degradation of compounds opens a large and promising market, attracting strong interest from companies - particularly those capable of turning pollutants into valuable products. This represents significant commercial potential, especially for bioremediation applications. No negative impacts are expected, as the project's focus on environmental cleanup and open-source accessibility provides clear benefits to the broader research community.

Based on this assessment, we made a deliberate decision to maintain CAPE as a fully open-source tool, rather than pursuing patent protection. This reflects our commitment to making environmental cleanup technologies accessible to researchers and organizations regardless of their resources. While we are currently focused on Rhodococcus opacus as our primary chassis organism, CAPE's architecture was designed to be highly scalable and extensible, allowing new functionalities and improvements to be added without major code revisions. This flexibility ensures adaptability to other chassis organisms and future metabolic engineering applications, keeping CAPE ready for potential integration in both academic and industrial contexts.

To sum up, HERO is an engineered Rhodococcus chassis for bioremediation, CAPE is a predictive tool for metabolic pathway analysis, and HELMET is a biosensor for quantifying TAGs. To build them, we formed a diverse team of biotechnologists, bioinformaticians and chemists. We also consulted numerous experts in biology, chemistry, bioremediation, and ethics, and engaged directly with water treatment plants and industry professionals. In the future, we plan to implement HERO in wastewater treatment systems, expand CAPE's predictive capabilities, and refine HELMET for practical use.

References

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Vázquez‐Boland, J. A., & Meijer, W. G. (2019). The pathogenic actinobacterium Rhodococcus equi: what's in a name?. Molecular microbiology, 112(1), 1-15.
Prescott, J. F. (1991). Rhodococcus equi: an animal and human pathogen. Clinical microbiology reviews, 4(1), 20-34.
Von Bargen, K., & Haas, A. (2009). Molecular and infection biology of the horse pathogen Rhodococcus equi. FEMS microbiology reviews, 33(5), 870-891.
Vazquez-Boland, J. A., Giguère, S., Hapeshi, A., MacArthur, I., Anastasi, E., & Valero-Rello, A. (2013). Rhodococcus equi: the many facets of a pathogenic actinomycete. Veterinary microbiology, 167(1-2), 9-33.
Eustace Y. Fernando (2025). Identification and recombinant expression of a novel defluorinase from Rhodococcus jostii RHA1, for defluorination and biotransformation of the PFAS compound 6:2 fluorotelomer carboxylic acid. Enzyme and Microbial Technology, Volume 191.
Alvarez, Héctor M., et al. "Formation of Intracytoplasmic Lipid Inclusions by Rhodococcus opacus Strain PD630." Applied and Environmental Microbiology, vol. 62, no. 5, 1996, pp. 1813–1818.