One of the most important of iGEM's values is the commitment to sharing contributions through open-source principles. During the creation and development of SuperBugBuster, our team was keen to think as much as possible about future iGEM teams. Our project sustainability lies in education, cooperation, and international solidarity. Our goal is to allow anybody to understand and take the most innovative elements of our project. These innovations are both scientific and non-scientific. On this page, we showcase all the parts of our project we offer to the IGEM community, hoping they will inspire even bigger projects than SuperBugBuster.
Many previous iGEM teams had worked on the use of CRISPR tools, but none had worked with a dCas9 fused to a cytidine deaminase to combat antibiotic resistance. Our team has documented the parts created in the Parts Registry. Together, these parts allow the bacterium to produce the dCas9 protein fused to a cytidine deaminase, which easily targets a particular gene. We have created the new BBa_K4818070 part containing a complete circuit with the TetR repressor, divergent and overlapping ptetR/ptetA as regulatory promoters controlling the expression of dCas9-Cytidine deaminase-uracil glycosylase inhibitor. This control circuit is inducible by anhydrotetracycline (Atc). In addition, this new part is flanked by attR1 and attL4 recombination sites and could be assembled by a multiplex LR Gateway reaction with other modules we have created, including the module carrying the specific gRNAs.
In addition, we created three other composites that, when added to the first, create a complete mutagenic plasmid. The construction of these parts is explained on the Experiments page and the Design page, while the parts can be found on the Parts page. We hope that our documentation will help future teams to use these parts as they see fit.
You can find more information about it on the Parts page.
In addition to a new part, our team has provided a direct application: facilitating the creation of a mutant bank. We have thus provided future teams with a new line of research. However, this part can also be used, for example, to study the expression of a gene in a bacterium and thus facilitate its characterization. As our composite part is constructed in the standard biobrick format, between restriction sites, it would be easy for anyone to extract it from our plasmid and insert it into their own. This would make it possible for any future team to study the expression of a gene or easily repress a resistance gene. In addition, they can use the software we have developed to create motifs to target the gene to be edited (as described below).
The use of a BacPROTAC system expands existing tools for targeting proteins specifically or even degrading them, so it will be interesting to see this used by future iGEM teams. PROTACs are a technology that has existed for over a decade, specifically targeting proteins to be degraded. Today, certain PROTACs that target proteins malfunctioning in cancer are now in Phase 3 of clinical trials. However, when it comes to BacPROTACs, things are quite different. Indeed, proof-of-concept for BacPROTACs was achieved in 2021 by Tim Clausen's team. This technology, similar to PROTACs, is different regarding proteasome recruitment since the latter differs between eukaryotic and bacterial cells. In our project, as well as being confronted with a bacterial proteasome with specific features, we also had to deal with a particularity of PROTACs: the synthesis of these molecules is done via chemistry.
However, we wanted to be able to make a BacPROTAC in plasmid form. So we had to change the structure of BacPROTAC so that it could be in gene form, and therefore protein form, in the bacteria. The changes are made on the part that targets the resistance protein. For that, we chose to use a Nanobody targeting OXA48. As for the region that binds to the bacterial proteasome, the simplest thing for us to do was to link the Nanobody to an optimally sized linker directly to the first multi-protein complex making up the bacterial proteasome. Thus, modifying the process and structure of (Bac)PROTACs to be used in plasmids and bacteria is innovative and may be useful for other teams wishing to use this technology for iGEM. We were keen to thoroughly document our research into this innovative tool for the specific degradation of proteins, as in the future, many iGEM teams may need to use it in the form of Biobrick.
You can find more information about it on the Experiments and Docking pages.
Our software is the first step in the design of our tool. Indeed, when constructing our plasmid and using CRISPR dcas9, we need guide RNAs that target the antibiotic-resistance gene but not the bacterial genome. In addition, as we are using CRISPR dcas9 fused to a cytidine deaminase, we need to ensure that this guide RNA can create a nonsense mutation. We have developed a real tool for selecting these guide RNAs, which did not exist until now. The available software could only find RNA guides one gene at a time due to the inability to input a complete database. That's the difference we've made! With our software, you can find everything at once.
This is very useful for finding guide RNAs that hit as many targets as possible, not just one. You can find out more about it on the Software wiki page. Our software is open source, which means that any scientist or iGEM team requiring assistance with their research can use it.
You can find more information about it on the Software page.
The creation of an all mathematical model on the use of our tool is a great innovation. Our model permits us to have an idea of how bacterias interact between them, how they transmit our plasmid and how they become non resistant to antibiotics. The code that builds our model is open source and online. Like this, scientists and other IGEM teams are able and free to reuse this code, to adapt it on their own biological tool. Indeed, we really wanted it to be accessible, to contribute in our field to the popularization of mathematics models that can be sometimes seen as really hard to understand. We tried as much as we could, to popularize our model, to be understandable easily. A representation of the model has been made, to also have a vision of it, and so that it is easier to understand.
Finally, to support this, we build an all tutorial, to help people to build their own model. Like this, we hope that mathematics and modeling would become more easy for people, and more easy to apprehend.
You can find more information about it on the Model page.
During our research journey, we used tools that helped us find what was best for our project. For example, for the BacPROTACs conception, we used some collaborative platforms carrying machine learning algorithms to select the best nanobody and linker. Due to the complexity of the algorithms, we created a manual to help future students use them with ease. RF Diffusion is a model for protein design. It has been created at Baker Lab, a renowned institute for in silico protein design. It uses structural information of many known bioactive compounds to create new compounds through machine learning algorithms. Compounds are ranked according to their bioactivity, allowing the best option to be chosen.
You can find the tutorial on the Predictions page.
To use this tool, it is possible to go on google collab.
The Onion's method is a methodological tool that was the basis for our human practices study. You can find it on our Human Practice page. Before SuperBugBuster, this method was still in development and had only been tested on digital tools. Firstly, the challenge was to adapt the method to a biological tool, which was of great interest to the creators (Celine Nguyen et Jean François Trégouët), and they were delighted by the result! The Onion can seem complex to execute, but once it's concluded, it gives us a highly organized and pertinent vision of the environment in which our tool is set. To facilitate its implementation for future teams, we created a tutorial that explains step by step the process. This method can now be applied to every iGEM project, allowing future teams to better understand the human and societal challenges surrounding their biotechnological tool!
You can find the onion’s method on the Integrated Human Practices page.
We all have the chance to take part in a unique and exciting experience based on a fundamental principle: collaboration. Synthetic biology is a field that continues to grow and evolve, and the challenges we face are complex and multidimensional. Collaboration in the iGEM competition is crucial in a number of ways. It enables us to draw on each participating team's varied expertise and skills. Each team member brings specific knowledge to the table, enriching our collective understanding and broadening our scope of possibilities. By working hand in hand, we are able to solve complex problems more effectively and innovatively. Collaboration within the iGEM community enables us to establish lasting links and create a global network of committed scientists.
By sharing our ideas, we strengthen our community and contribute to the growth of synthetic biology as a whole. Together, we have the power to push back the boundaries of science and make a real difference in the world. With our collaboration with Goethe Uni, we realized that other projects could be complementary to ours. Like that by collaborating together, we could have some real opportunities to change the game. Our collaboration about antibiotic resistance was also a real benefit to our project, as some teams answered some questions in some direction we had yet to consider. Lastly, collaborations like the mascot collaboration and the postcard project from iGEM Düsseldorf enabled us to discover other high-quality iGEM projects and maybe inspire us for the future.
You can find more information about it on the Collaboration page.
The education team also worked for the community by creating a card game. The education part of our project was essential to us, as it is the core of the antibiotic resistance problem. We created a game based on the Werewolves of Miller's Hollow, a well-known card game in France. The game's design and rule adaptation allow for an easy understanding of antibiotic resistance, how to fight it, and the differences between bacterial and viral infections. It was gifted to our crowdfunding donors and those with whom we tried our game. Besides being adapted for educational institutions, our game can also be played in families, libraries for children, or during summer camps.
We also created an all set of posters for sensibilization purposes about antibiotics and their good use. Indeed, we have in total five posters, realized by our team. This sensitization campaign is a valuable tool for educating people on proper antibiotic use. We use flyers, posters, and different mottos to impact more people. Our goal when designing this campaign was to envision it in pharmacies, doctor's offices, hospitals, schools... Basically anywhere appropriate to educate the population.
You can find more information about it on the Education page.
In today's digital age, effective communication through social media has become an indispensable tool for iGEM projects. It is not just about scientific innovation but also about disseminating knowledge, promoting inclusivity, and addressing global issues that often go unnoticed. Social media platforms provide the means to disseminate research, methodologies, and results to a global audience. Doing so can inspire others to get involved in synthetic biology and interdisciplinary scientific projects. iGEM projects often involve complex technical aspects. Social media offers a unique opportunity to demystify these complexities and make them accessible to a broader audience. Teams can use engaging visuals, infographics, and user-friendly language to break down intricate scientific concepts, ensuring that even non-experts can grasp the significance of their work. Social media platforms are also a powerful medium for promoting inclusivity within the scientific community. This inclusivity aligns with iGEM's commitment to diversity and equality. Many iGEM projects address pressing global challenges, such as health crises, environmental issues, and food security. Social media allows us to shine a spotlight on these problems and the innovative solutions they are developing. By sharing their insights and findings, we can raise awareness about critical issues that require immediate attention. So, remember, in the world of iGEM, social media is not just a tool; it's a catalyst for change and progress!
You can find more information about it on the Communication page.