Team:Evry Paris-Saclay/Human Practices

Evry-Saclay 2021 iGEM team website

Human Practices

Our planet and society are nowadays confronted with multiple new issues: antibiotic resistance, feeding the planet, invasive species, emergence of new viruses or micro-organisms harmful to crops… So many challenging problems!

The Human Practices section has allowed our team to consider many of these societal issues. Here, we will explain how we set up our strategy to question the feasibility and responsibility of our project from the point of view of the scientific community, global health, and our planet.

The iGEM Evry Paris-Saclay 2021 team has developed a tool to direct the evolution of nucleic acid sequences to generate new proteins with novel properties. It is, therefore, necessary to question certain aspects of our directed mutagenesis project, such as the risks that it raised, or the benefits that this new technology can bring. To answer these questions from a legislative point of view, we have relied on various publications that we will discuss later (legislation on directed mutagenesis [1, 2, 4, 5, 7], risks and benefits of this technology [6]). This allows us to evaluate the viability of our project, and see its feasibility in the future.

The main objective is to take into account and respect all of these aspects, bringing our project to its highest level of efficiency and potential. Indeed, discussions with various experts, working in protein modeling and engineering, have helped us to design a more efficient and ethical project.

Bioethics

The new genes and proteins that will be generated by directed evolution must be regulated by legislation to avoid any undesirable use. Acceptability of the various stakeholders, especially the population, with whom information and awareness-raising work must be carried out so that the technology is understood and how it should be used according to the various legislations.

We have consulted the French Senate Report [1], in order to understand the legislation around directed mutagenesis, which allows us to understand the limits of the application of the process for environmental or medical purposes. Our project, being based on the random modification of nucleic acid sequences, the only limit is that we have no control over the type of proteins that would be generated, nor in its danger and its environmental and sanitary impact, or their usefulness for medical applications, or biotechnology.

The current problem is the feasibility of the project within the European Union, in accordance with European legislation, which is placed above national laws. In France, several laws exist on bioethics. For example, law n° 2004-800 of August 6, 2004 relating to bioethics, law n° 94-548 of July 1, 1994 relating to the processing of nominative data for the purpose of research in the field of health and modifying law n° 78-17 of January 6, 1978 relating to data processing, files and freedoms, law n° 94-653 of July 29, 1994 relating to the respect of the human body; - Act No. 94-654 of 29 July 1994 on the donation and use of elements and products of the human body, on medically assisted procreation and on prenatal diagnosis [1].

For instance, European legislation is stricter than that in the USA. Indeed, in the USA, genetically modified organisms (GMO) are less tightly regulated [2] as compared to the European Union legislation, which considers organisms as GMO if genome editing was used, but not when chemical and radiation is used to induce random mutagenesis [3]. The stricter legal framework can be especially challenging for the agri-farming sector in disallowing GMO technology to limit losses due to disease or bad weather, for example, and therefore partly preventing the progress of research in this field, particularly for the fortification of plant genomes. As a result, the EU could lag behind China and the USA in innovation, which could have a significant adverse impact on the European agricultural sector [4,5]. The “gap of law” is also a huge challenge for scientists. Indeed, discoveries are going fast and the law needs to be reformed in a timely manner to answer new cases issues.

Environmental and sanitary considerations

Our project aims to be safe for human, animal, and plant populations, and to be as non-polluting as possible. Directed evolution can be a very interesting tool, but it must be used responsibly. Our Evolution.T7 directed evolution system may be dangerous if it evolves an undesirable trait whether in the laboratory or in industrial settings. Indeed, the risks associated with directed mutagenesis are multiple, such as the lack of knowledge of off-target effects for the organism or the ecosystem, and the lack of traceability for gene editing events. The lack of anticipation is linked to the misuse of the technology and unintended consequences that cannot be predicted.

Sustainable Development Impact

On a broader scale, the goal of our project is to establish a method for directed evolution, bringing a tool to develop products and applications sustainably. Our project aligns with concepts related to sustainable development goals, but especially regarding innovation. The key feature of our project is to bring a modular and efficient system for directed evolution applications. Having this point in mind, our project related to the efforts to lean to an economic growth associated with sustainable production, in this case, based on biological systems used sustainably.

On our own scale, we are trying to provide, through our method, new sustainable solutions, especially in terms of sustainable development. But how can our project be beneficial for sustainable development? The applications of directed mutagenesis are multiple:

  • Medical/therapeutic applications
  • Agricultural applications
  • Environmental applications

In the therapeutic field, site-directed mutagenesis would make it possible to increase the effectiveness of vaccines, which in the long term would lead to the eradication of infectious diseases. But also to find biomedicines against certain diseases, such as malaria. Directed mutagenesis would therefore make it possible to eradicate the disease by targeting the genes responsible for the resistance of the parasites in the mosquito. It would make it possible to improve certain therapies, particularly gene and cell therapy, in order to treat genetic diseases. These techniques of directed mutagenesis are only applicable to somatic cells, as it is unethical to modify the genome of germ cells.

As far as agriculture is concerned, directed mutagenesis would meet the challenges of sustainable development through the various solutions it could provide. It would provide solutions for increasing production, reducing labor, and reducing the amount of gas emitted into the atmosphere by crops, by targeting the genes responsible for root growth, for example. But also, to increase the resistance of crops to diseases, especially those brought by vectors such as parasites, which can destroy an entire crop, or even restore the diversity lost due to the domestication of certain plants and increase the reproducibility of crops.

The different sustainable development goals can be tackled by evolving the appropriate proteins on a case by case basis.

Economic aspects

For this tool to be usable for industrial purposes, the total cost must be as low as possible. This process would generate a library of proteins and nucleic acid sequences for scientific and industrial use. Directed evolution offers a cost-effective solution in the R&D sector.

Safety and Security

Self-replicating re-engineered cells may produce undesired consequences if they escape or overwhelm their intended environment. Some mechanisms could be used to prevent the transfer of a plasmid from engineered host bacteria to wild-type. Indeed biological systems that do not interact with those occurring in nature or to a very limited extent would be the solution. We could think about several points: physical containment, such as microencapsulation, or making no advantage for the environment to uptake such a system (auxotrophic selection). The inability to replicate (conditional plasmid replication) and the incorrect translation (recoded tRNA use) could be a solution, as well as no host immunity (restrictions enzymes immunity) or endogenous toxicity (Sok, toxin-antitoxin system). Thinking about an orthogonal system, which are biological systems that do not interact with those occurring in nature or to a very limited extent, was necessary. With the help of different professors, we were able to discuss different aspects of such systems (see below).

As our project´s proof of concept validation depended on antibiotic selection, all considerations for handling the compounds were followed. First, the characterization was performed in a non-pathogenic E. coli strain, and the antibiotic used was a laboratory standard. The directed evolution validation succeeded with a classic antibiotic resistance phenotype, and by using a standard laboratory procedure to validate evolution-related systems. Yet, despite the importance of studying antibiotic resistance genes, E. coli is still, in broader terms, an infection-related strain. Considering this fact, good lab practices were at the center of our wet lab team while developing and using our system.

Inclusivity and more

At iGEM Evry Paris-Saclay Team 2021, everyone is welcome to discuss science in a friendly and welcoming environment. Indeed, our team is rich in colors and experiences, being composed of elements from all horizons in terms of origins, ages, and fields of study (biology, computer science, engineering). More than a desire to share our passion for synthetic biology, we want to convey the message of a universal community where everyone should feel at home and confident to share their ideas, in the respect of all. The love of science can only be revealed and develop its full potential within teamwork. In this respect, iGEM has taught us much more about the fantastic world of synthetic biology, but also patience, tenacity, and listening to others. These values have been called upon many times, having had numerous discussions with several teams around the world. Indeed, while looking for collaborations, we realized that sharing our project with other teams was a good way to create an open and welcoming scientific environment. The French meet-up that we co-organized allowed us to have a good time with different European teams. We presented our project at the Cité des Sciences et de l'Industrie during the Fête de la Science in Paris, which allowed us to meet scientists, children, and all those curious about synthetic biology, to share our views on the societal implications of research. By participating in this event, we wanted to allow more people to contribute and participate in our project. For our project on the theme of the directed evolution of proteins, we wanted to discuss with professionals in various fields such as law, philosophy, or sociology for example to bring us elements that would be interesting to consider in relation to our project.

Our wish was also to post some advice from researchers working on directed evolution, on the same model as the TUDelft 2019 team, by making a list of interesting advice and remarks that they would have given us in order to compare with existing evolution techniques, or even to confirm that our directed evolution tool is interesting and could be useful if it was developed in such or such way.

Time to discuss! Our Interviewers...

Our team had the great opportunity to discuss with professors and researchers in various fields, to be able to build our project and answer key problematic questions.

We had an interview with Professor Dr. Phillipe Minard, who works on modeling and protein engineering at the Institute for Integrative Cell Biology at Paris-Saclay University, to develop the most suitable and efficient directed evolution tool possible. We talk about screening, selection of mutants, how to get our mutated genes, and to better understand the process of directed mutagenesis. The main points we had to take into account in our project:

  • Method of selecting proteins with nanobodies, if one wishes to obtain proteins for therapeutic purposes, or scaffold proteins if it is for biotechnological purposes
  • Screening by resistance to proteases
  • To perform a bacterial display, and recover the gene that codes it, it is necessary to expose these elements to the surface of bacteria
  • Taking into account the mutational load
  • The target depends on the selection, so the objective
  • Interaction by antibodies or scaffold proteins

Dr. Stegan Wolfl, Professor in Pharmaceutical Biology, Bioanalytics and Molecular Biology, at the Ruprecht-Karls-Universität Heidelberg helped us to consider the idea of an orthogonal replication system, as you can see below:

Dr. Stephen Davil Bell, molecular and cellular biochemistry Professor at the Indiana University Bloomington in the United States, has also supported this idea of orthogonal replisomes among a really interesting discussion.

Dr. Mario Mencía, Professor at the University of Madrid and researcher at the Molecular Biology Center “Severo Ochoa '' helped us as we were choosing his system as a starting idea point for our project. We were able to talk about many things such as the mutagenic factor, indel generation, or deaminases. He helped us to highlight some key points of our project such as what enzyme we should attach to the T7RNAP, or what mutator domains other than deaminases could be interesting in our case study. We also discussed the targeted aspects of our tool and nanobodies.

We contacted Dr. Murphy Michael to forward our inquiry to Dr. Roccor Raphael who was able to help us. We discussed orthogonal replication systems to generate the mutations, the idea of adding a transposon part in the process to only diversify the structural protein gene and not other places that we did not want to mutate like the promoter and the screening design.

We also contacted Dr. Brendan Hussey who advised us on some techniques and papers.

We had a discussion with Dr. Serge Muyldermans, an expert in nanobodies and structural biology at Vib-Vub Center for structural biology, about the way to generate our mutants and the affinity maturation by directed evolution.

Finally, Dr. Patrice Courvalin (Institut Pasteur Unité des Agents Antibactérien), antimicrobial resistance expert, and a medical doctor of the hospital Edouard Herriot of Lyon, helped us with practical aspects of the β-lactam antimicrobial resistance in the hospital.

We were glad to have the opportunity to discuss with all these professionals and experts and would like to thank them.

Conclusion

In summary, our project will bring us solutions for the future: to fight against the loss of agricultural yields, the disappearance of endemic species, or allow progress in the therapeutic field. The technologies used in genome editing, of which our project is part, must be used responsibly, in order to avoid all possible risks or side effects linked to the manipulation of these technologies. The different meet-ups and discussions in which our team has participated have allowed us to discuss synthetic biology and other subjects with various teams and professionals from different fields.

References

[1] French Senate report "The economic, environmental, health and ethical challenges of biotechnologies in the light of new research avenues" on 14 April 2017

[2] Eriksson D, Kershen D, Nepomuceno A, Pogson BJ, Prieto H, Purnhagen K, et al. A comparison of the EU regulatory approach to directed mutagenesis with that of other jurisdictions, consequences for international trade and potential steps forward. New Phytol. 2019 Jun;222(4):1673–84.

[3] Van Der Meer P, Angenon G, Bergmans H, Buhk HJ, Callebaut S, Chamon M, et al. The Status under EU Law of Organisms Developed through Novel Genomic Techniques. Eur J Risk Regul. 2021 Jan 6;1–20.

[4] Déclaration du groupe des conseillers scientifiques principaux, Commission européenne “Une perspective scientifique sur le statut réglementaire des produits dérivés de l’édition génomique et ses implications pour la directive OGM” publiée le 13 novembre 2018

[5] Regnault Roger C. “Biotechnologies d’édition du génome : l’UEAA demande une modification de la réglementation européenne”- European Scientists. 09 November 2020

[6] Shukla-Jones A, Friedrichs S, Winickoff D (2018), "Gene editing in an international context : Scientific, economic and social issues across sectors", Documents de travail de l'OCDE sur la science, la technologie et l'industrie, n° 2018/04, Éditions OCDE, Paris.


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