Bologna - iGEM 2025

Abstract

In the 21st century, the face of remediation is changing, and biotechnological solutions are increasingly replacing traditional chemical and physical methods, offering safer and more sustainable alternatives. Indeed, in bioremediation – the degradation of pollutants using living organisms – there are many bacterial strains reported to degrade pollutants and a vast body of literature describing them. However, what is often missing to make biology a true engineering discipline—one that is accurate, reproducible, and controlled—are a standardized methodology and a set of model organisms.

HERO's tale

Hi, there we are here to tell you a special story, the story of how HERO was born...

Well, it all started in Bologna, where we were trying to tackle the growing problem of pollution in our oceans:

While digging through mountains of scientific literature for a solution, we stumbled upon a paper from Japan. It described a unique bacterium, Rhodococcus oxybenzonivorans, with an incredible ability: it could essentially eat BP3, breaking it down into harmless compounds.

At first, we were ecstatic. "This is it! The perfect answer!" But we hit a wall almost immediately. How were we supposed to work with a bacterium that was not only thousands of miles away, but was also a complete mystery to science? It was almost unusable for us.

Then, sitting around and brainstorming, an idea sparked: "What if we don't need the entire bacterium? What if we could just borrow its superpower?"

Our plan became clear: we would identify the specific genes responsible for degrading BP3 and transfer them into a reliable, well-understood bacterium that we use in the lab every day.

But that brilliant idea just opened a floodgate of new questions for us. Which bacterium would be the best new host? And more importantly, out of all the genes in the predicted cluster, which ones were the essential keys to the whole process?

That was our turning point. We realized we needed to think bigger. We needed more than just a single solution for a single problem. What we really needed were better tools for the job. We needed a highly customizable bacterial platform that could be easily engineered for new tasks, and a powerful predictive tool that could analyze gene clusters and tell us which parts were important.

And that is how our journey with HERO began.

Introduction

The genus Rhodococcus represents a phylogenetically and catabolically diverse group of Gram-positive, non-motile, aerobic bacteria (Bell et al, 1998).

The peculiarities of the members of this genus are their ability to utilize a wide variety of carbon and energy sources (Ludwig et al., 2012) and their extreme and contaminated isolation environments (Kim et al., 2021; Xiang et al., 2022). These traits are strongly connected to one another: their global distribution and ecological success are directly linked to their unique environmental persistence and robustness (LeBlanc et al. 2008; Cappelletti et al. 2016).

Here are some examples of Rhodococcus strains - hover over each card to discover which pollutants they can degrade!

Rhodococcus sp. 14

PAHs

Polycyclic Aromatic Hydrocarbons

(Song et al. 2011)

Rhodococcus ruber IEGM 231

Crude Oil

Petroleum compounds

(Ivshina et al. 2016)

Rhodococcus rhodochrous BX2

Aliphatic Nitriles

N-containing compounds

(Fang et al. 2015)

Rhodococcus opacus 3D

Phenol

Aromatic compounds

(Anokhina et al. 2025)

Rhodococcus pyridinivorans P23

PET

Plastic polymers

(Guo et al. 2023)

But, what's the Missing Piece?

Despite the incredible potential of this genus, we have to recognize the challenges ahead. Many Rhodococcus strains have been identified as efficient pollutant degraders, but only a few are currently used in industry due to the limited availability of reliable genetic parts and engineering tools (de Lorenzo et al., 2018). To cover this "lab-to-market gap," we need strains that are not only powerful degraders, but also economically feasible, predictable, robust, safe, and easy to engineer. Many naturally promising strains fail to meet these strict requirements.

To address this challenge, we are developing a new chassis for bioremediation based on Rhodococcus opacus PD630. This strain will serve as a customizable platform for the integration of degradation genes from other Rhodococcus species, ultimately making the engineering process cheaper, faster, and more sustainable.

HERO project

To address the missing piece, we designed a next-generation chassis strain of Rhodococcus opacus PD630.

1. Integration of a Recombinase into the Genome

Our master strain is engineered to stably express a site-specific recombinase (see Project Design page), which acts as the key player during the insertion of new DNA fragments—whether they are uncharacterized sequences or degradation pathways.

Fig 1: The genome of Rhodococcus opacus PD630 that has become HERO after the insertion of Cre/lox system. TipAL is the transcriptional regulator protein that controls the pTipA promoter. ThioR is the gene for the resistance to Thiostreptone.

2. A Plasmid Vector for Seamless Recombination

We developed a specialized plasmid carrying lox sites, which can recombine with the pre-inserted genomic locus under the control of Cre recombinase. This provides a straightforward and predictable way to add new functionalities.
So that is how it works:

Fig 2: insertion of degradation genes inside HERO genome.

3. Promoter Characterization for Predictable Gene Expression

To establish R. opacus as a robust chassis organism, a reliable set of promoters is essential. Our team is currently testing a toolkit of four different promoters: the common pLac promoter, and three variants derived from a native Rhodococcus jostii RHA1 promoter characterized by Round et al. (2019). This toolkit will ensure predictable, tunable, and standardized expression for future applications.

By combining a recombinase-based integration system, a versatile plasmid vector, and a library of well-characterized promoters, we aim to provide the synthetic biology community with a promising modular chassis for bioremediation.

CAPE tool

We developed CAPE as a user-friendly platform to support researchers in the design of metabolic pathways for biodegradation in Rhodococcus opacus PD630.

1. Predicting Biodegradation Pathways

Starting from a source and a sink compounds as inputs, our tool identifies possible metabolic routes by combining curated KEGG database information with predictive softwares capable of filling missing metabolic links.

2. Selecting and Characterizing Enzymes

For each predicted reaction, CAPE suggests candidate enzymes and retrieves their protein sequences from public databases. This step helps users explore multiple enzymatic options and tailor the pathway design to their needs.

3. Optimizing for Expression in Rhodococcus opacus PD630

We implemented a codon optimization system based on the R. opacus codon usage table, ensuring that all selected enzymes can be efficiently expressed in our chassis. CAPE also checks for restriction sites incompatible with iGEM assembly standards (or custom ones), simplifying downstream cloning.

By integrating pathway prediction, enzyme selection, and sequence optimization into a single modular pipeline, we created CAPE as a user-friendly platform that empowers teams to move from conceptual pathway design to assembly-ready genetic parts with minimal effort.

Fig 3: workflow of CAPE tool.

HELMET biosensor

We are engineering HELMET, a colorimetric biosensor based on an enzymatic cascade initiated by lipase (Pohanka, M., 2019). The system is designed to detect triacylglycerols (TAGs) (Kubo, A. et al., 2013), (Tarvainen, M. et al., 2019), which R. opacus accumulates as a metabolic products. The lipase hydrolysis releases intermediates that feed into a downstream enzymatic cascade, ultimately producing a visible colorimetric signal proportional to TAG concentration (Ramesh, M. et al., 2022), (Zhai, Q., et al., 2019). This approach provides a specific, low-cost, and field-deployable detection platform, bridging synthetic biology with environmental monitoring and bioprocess control (Ramesh, M. et al., 2022).

Fig 4: workflow for the TAGs colorimetric biosensor.

A sample is added with controls, then goes to the PPL wells for an enzymatic reaction; then the resulting solution moves to the MR wells, creating a color signal if TAGs are present. A positive result shows a color change, while a negative result does not.

Facing Problems

Stable Chromosomal Integration

Unlike plasmid-based expression, which requires constant antibiotic selection to be maintained, this system integrates genes directly into the bacterial chromosome. This ensures that the new genetic circuit is stably maintained and faithfully inherited by daughter cells, leading to more reliable and consistent strain performance.

Elimination of Antibiotic Resistance Plasmids

After the gene of interest is integrated, no origin of replication is available in Rhodococcus. The final engineered strain therefore presents antibiotic-resistance genes only inside the genome and not on a plasmid, which minimizes the risk of spreading resistance genes via horizontal gene transfer. Furthermore, selective pressure is not required, allowing application in more environments without antibiotics use.

Standardized and Efficient Workflow

This system creates a customized platform for engineering Rhodococcus opacus. Once the master strain containing the inducible Cre recombinase is built, inserting any new gene becomes a routine process:
1. Clone the gene of interest into the standardized delivery plasmid.
2. Introduce the plasmid into the master strain.
3. Induce Cre expression.
4. Select recombinant strains.

This standardized workflow increases the speed, efficiency, and reproducibility of genomic modifications.

Precise and Predictable Integration

The Cre-lox system is site-specific, meaning the new gene is inserted into a single, predetermined location in the genome. This avoids the risks associated with random integration, such as disrupting essential native genes or causing unpredictable expression due to "position effects." This precision leads to highly predictable outcomes and functionally consistent clones.

High Versatility

HERO is a versatile chassis that can be used to integrate a wide variety of genetic payloads. Whether you're inserting a single gene, a complex metabolic pathway, or a sensor circuit, the core integration method remains the same. This makes the platform highly adaptable for future projects and diverse applications.

Predicting new metabolic pathways

Nature already offers a large variety of Rhodococcus strains with specialized degradation abilities. The problem is connecting the knowledge that we have with the exact pathway that can convert a pollutant into a carbon source. This is why CAPE was born.

Ready-to-use TAG Biosensors

With HELMET, we are working on a simple, rapid, and low-cost reporter system to monitor TAGs production. These biosensors will serve as practical tools for both laboratory testing and field applications.

If you want to know more about what HERO was, is and will be, go and take a look at the Human Practices page! 👀

To Sum Up

Our aim is to empower researchers in bioremediation by providing:

References

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De Carvalho, C. C., Costa, S. S., Fernandes, P., Couto, I., & Viveiros, M. (2014). Membrane transport systems and the biodegradation potential and pathogenicity of genus Rhodococcus. Frontiers in physiology, 5, 133.
Lee, S. D., & Kim, I. S. (2021). Rhodococcus spelaei sp. nov., isolated from a cave, and proposals that Rhodococcus biphenylivorans is a later synonym of Rhodococcus pyridinivorans, Rhodococcus qingshengii and Rhodococcus baikonurensis are later synonyms of Rhodococcus erythropolis, and Rhodococcus percolatus and Rhodococcus imtechensis are later synonyms of Rhodococcus opacus. International Journal of Systematic and Evolutionary Microbiology, 71(7), 004890.
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