Superbugbuster, a project by the iGEM insa-ens-Lyon1 team!

As part of our partnership with iGEM, the biggest synthetic biology competition in the world, we are showcasing some of the exciting projects from teams that share our vision of synbio impacting positively on human health. In this first post, we've let the iGEM team from Lyon take over our blog to share what they are working on. In this blog you will learn about the issue of antibiotic resistance that the team have decided to work on, how they have assembled an interdisciplinary group of experts and how they are using computer programming to identify RNA to solve the problem.

The problem we want to solve

Imagine a future where we could die from a harmless infection. This time is now closer than we think, due to a potent function developed by bacteria: antibiotic resistance.

Since the discovery of antibiotics in the 1920s, they have been widely used in our society and have since undergone significant development. Their consumption has become excessive and poorly regulated. Due to this, certain bacteria have been subjected to selection pressure, driving them to develop resistance genes against antibiotics. Fewer and fewer treatments are now effective, and an increasing number of bacteria are becoming resistant; we are heading toward a deadlock. Antibiotic resistance is responsible for over a million deaths each year worldwide and is projected to be responsible for over 50 million deaths annually by 2050. It is a scourge that requires immediate action!

This is the issue that our team - iGEM insa-ens-lyon 1 - has focused on for the iGEM 2023 competition. Like all iGEM projects, our approach to the subject consists of several parts: a purely biological element to develop a tool to combat antibiotic resistance, a computer program to assist us in designing the tool and ensuring its safety, and a human-practices aspect that enables us to grasp and understand the implications of our project while raising awareness about this issue, which is still too often overlooked. The goal is to re-sensitise bacteria that have become resistant to antibiotics. 

Step one - building an interdisciplinary team!

To achieve this, we gathered a team consisting of students from three universities in Lyon: INSA, ENS, and the University of Lyon 1. Leading our team is Camille Bacquié, our team leader, and our project wouldn't have come to fruition without our Principal Investigator, Sylvie Reverchon, a microbiology professor at INSA Lyon. Speaking of biology, Adel Amine Gani and Jade Bonnelle take the lead, with valuable assistance from Arthur Loup, Félicités Rapon, and Maëlys Alazard. The development of our computer program wouldn't have been possible without the contributions of Arnaud Mengelle and Quentin Duvert, the latter being the most versatile member of our team. Handling human practices and education, Léa Aguilhon and Natacha Doutreleau lead the way. Timothée Massot and Clarisse Masse serve as our logistical experts, enabling us to realise our boldest dreams. Lastly, Andie Beaubiat leads the project's communication efforts and infuses it with creativity as the team's graphic designer.

As our diverse team of students from Lyon's INSA, ENS, and the University of Lyon 1 came together to tackle this challenge, we harnessed a broad range of expertise. From molecular biology to bioinformatics, our collaboration allowed us to develop innovative solutions for fighting antibiotic resistance.

Tools and techniques

For the design of our tool, we have chosen to employ two different techniques: the CRISPR system and the BACPROTAC system.

CRISPR is the famous molecular scissors technique. Using a cytidine deaminase module, guide RNAs lead the system to our gene of interest, the gene responsible for antibiotic resistance. A codon is modified by a nonsense codon in the middle of these genes, thanks to guide RNAs that are specific to them, leading to the inactivation of the resistance genes. After transcription and translation, the result is a non-functional protein, leading to the loss of resistance.

On the other hand, the BACPROTAC system is relatively new. It involves a small molecule capable of binding to a protein we want to eliminate, a protein responsible for antibiotic resistance in our case. It also binds to the bacterial protein degradation system on the other end. As a result, the targeted proteins are removed, making the bacteria sensitive again to antibiotics.

By targeting both resistance genes and proteins, our tool provides a comprehensive approach and proves to be a promising system in the fight against antibiotic resistance. To ensure the safety and proper functioning of our tool, we rely on a computer program that we have developed. This program enables us to identify all guide RNAs that can bind to the target sequence upon which the CRISPR system must act.

We begin with a database containing the genes we want to deactivate. The software then scans this database to locate all the patterns we have identified as potential guide RNAs: 20-nucleotide motifs ending with the sequence NGG. Subsequently, we cross-reference our results with the database of genes within our bacterium, which serves as the carrier for our system. We must ensure that our tool will act exclusively on the genes we wish to deactivate and not on the basic genes present in the bacterium to avoid unintended mutations. Therefore, the software checks if each previously identified motif exists in the bacterium's genome and removes everything found there. This yields the final list of guide RNAs that we can use, and the next step is to select the ones most suitable for functioning with the cytidine deaminase system to induce stop codons.

The aim and the use of the program 

Our aim was to create the most versatile software possible. Indeed, it can be generalised for searching motifs for any CRISPR system and for any initial database. Consequently, our program could become a highly valuable tool for synthetic biology in the long run. As we delve into the features of this software, we also recognise the importance of responsible technology development. This brings us to our discussion of human practices, which is also a big part of the iGEM project.

The approach and the concept 

Our approach initially involved examining the causes and consequences of antibiotic resistance in society. We contacted numerous experts in the field to address our questions and guide our thinking. In each interview, a recurring theme emerged: Antibiotic resistance is a global phenomenon that affects not only human health but also animals and the environment. It was crucial to adopt a holistic perspective that encompassed these three dimensions in our deliberations, leading us to focus on the concept of "One Health".

Our team also took the time to contemplate the future of our tool, particularly how it could be adapted for use in humans. We believe that our system could serve as a complementary treatment, possibly in the form of a probiotic, allowing it to have an impact on bacteria that have become resistant. However, this is not without risks, and it is imperative to consider various scenarios and ensure the safety of our project.

By doing so, we aim to address the primary goal of iGEM, which is to tackle a global issue through biotechnology and synthetic biology. We hope that our project will raise awareness about the phenomenon of antibiotic resistance, thanks to our promising tools and communication efforts aimed at reaching a broad audience. One of iGEM's core values is sharing and collaboration, and we have had the opportunity to meet and collaborate with other equally brilliant teams along the way.

In envisioning a future where antibiotics remain effective, the SUPERBUGBUSTER project stands as a beacon of hope. Our mission is clear: to fight antibiotic resistance on a global scale, protecting not only human health but also the well-being of our environment and all living beings.