Team:GO Paris-Saclay/Human Practices
INTEGRATED HUMAN PRACTICES
In this changing world, the promise of synthetic biology is to improve the sustainability of our actions. Helping to feed the world, caring for people, and finally using healthy and renewable energy. It is about reinventing the world we know towards a model closer to nature, to make it sustainable and prosperous. One solution is the use of biological systems, to produce in a more sustainable way. Indeed, we can get inspired by nature to create biobased products more simply. By 2025, the biobased chemical sales are expected to grow about 15%, and therefore will take more and more relevance in our future.
Building GEMs that are genetically more stable
At the onset of the project was an interest in constructing genetically engineered machines (GEM) that would be genetically more stable: synthetic genetic circuits integrated in a chassis would be less likely to mutate and become non-functional if they were entangled with components essential for chassis multiplication. The prospect of entangling a biobrick with an essential gene in a chassis seemed like a simple and good idea that would be useful to synthetic biologists.
For our Worldwide virtual Meetup, we were lucky enough to welcome a former iGEMer, who is now an engineer. Maher BEN KHALED is a graduate student at the Toulouse Biotechnology Institute at the INRAE (National Research Institute for Agriculture, Food and Environment). His project deals with the biodegradation of plastics. Our conversation with him allowed us to realize the importance that our project could have in the use of GEMs, used on an industrial scale, and thus helped us to deepen our reflection on this aspect.
Indeed, many industries that use GEMs, including food, pharmaceutical, cosmetics and biofuels industries, are faced with the problem of the stability of their genetic construction. All these companies manufacture biomolecules from micro-organisms grown in fermenters.
For instance, the production of the antibiotic erythromycin is carried out using GEMs but poses problems of maintaining the production genes. Despite forced maintenance by selection on antibiotics or alternative strategies, there is still a loss of plasmid in 10% of strains, and most importantly erythromycin production becomes highly variable. (Fang et al, 2017)
Concerning the production of 3rd generation biofuels, the same problem arises with cyanobacteria modified to produce biofuel. Cyanobacteria have a machinery for inactivating artificially introduced genes, which poses the problem of maintaining the production system over time, and therefore the profitability of production. Moreover, the biocontainment of algae in reactors is another crucial issue. (Rosenberget al, 2008)
Getting a scientist involved in metabolic engineering to adopt genetic entanglement and designing some HugGenesS for him to test
The production of many biomolecules requires metabolic engineering to make it economically more viable and thus more attractive. In order to get closer to this optimization of GEMs for industrial use, we asked a researcher working on optimising the metabolic pathways for lipid production to give us some of his time.
Dr Tristan Rossignol is a researcher at INRAE who works on lipid production in yeast (Czerwiec et al, 2018). He is actually a work package leader in the European project CHASSY (Model-Based Construction and Optimisation of Versatile Chassis Yeast Strains for Production of Valuable Lipid and Aromatic Compounds). As the optimisation of metabolic pathways is at the heart of Tristan Rossignol's work, our interview addressed the most concrete aspects of the stabilisation of exogenous genes. More than an interview, we managed to establish a real work exchange with Dr Tristan Rossignol. Following this discussion, Tristan Rossignol came back to us, asking us to tangle two genes for his model organism Yarrowia lipolytica. We generated a series of HuGenesS to interweave genes encoding fluorecence proteins with genes conferring antibiotic resistance (listed in the engineering success page). He ordered DNA fragments and is currently cloning some of our designs. Stay tuned!
Bioconfinement at the time of the global spread of a virus
At this point, we started wondering if genetic stability was the only problem to solve. Indeed, industries producing biomolecules in fermenters use GEM microbes that have been optimized to withstand the fitness burden associated with the production, but these microbes are also genetically modified organisms (GMOs) that should not be released into the environment, on purpose or by accident. In fact, genetically modified microorganisms, whatever the nature of the modification, are designed in a strict environment with a specific purpose. The modifications created could become harmful or inappropriate if the microorganism were to change its environment and thus evolve in a natural setting. We have no certainty that they will be harmless to humans and the environment. Could genetic entanglement be useful in the biocontainment of genetically modified organisms (GMOs)?
In March, we had the opportunity to physically meet the Marburg Team, where we discussed our projects and past experiences at length. But, above all, we were students interested in biology in the context of the emergence of a new virus. And it was during the discussions with the German team that we were able to become aware together of the reality and the extent of the damage that can be caused by the spread of a small virus, and therefore of the importance of biological containment. Because our project is not alien to our global health situation, we all seek to live in a safe and sustainable world, where man can live in harmony with the rest of the world without being the cause of his own loss. This meeting became part of our HuGenesS project thanks to the very idea that emerged that evening: the idea of a containment that has been realised in our countries, as in our project.
The 2020 sanitary context reminded us that containment remains a crucial issue for GEMs (Torres et al, 2016). Many methods have been developed with the aim of confining these organisms, such as addiction strategies or even kill-switches to self-destroy, without finding a miracle solution (Simon et al, 20016:).
Understanding the laws and regulation around GMOs in Europe
In order to better understand the political and social issues GMOs have to face, we took a look at the legislation in France and in Europe about GMOs. Because the reluctance (or fear) of the general public, and because the Schengen area allows the free movement of products, the legislation is very well described in the texts of law.
The French legal definition of GMOs is given in the Environmental Code as follows:
Art. L. 531-1: "Genetically modified organism: organism - any non-cellular, cellular or multicellular biological entity capable of replication or of transferring genetic material; this definition includes micro-organisms, including viruses, viroids and plant and animal cell cultures - whose genetic material has been modified otherwise than by natural multiplication or recombination."
European legalisation allows the "contained" use of GMOs "in particular for research, development, teaching or industrial production purposes" (Art. L. 532-2) as long as this use is made "in a way that respects the environment and public health, agricultural structures, local ecosystems and production and commercial chains qualified as 'genetically modified organism-free', and in complete transparency. "(Art. L. 531-2-1).
This contained use is subject to the approval of the High Council for Biotechnology, in order to comply with the rules published above. If, moreover, the use is made under conditions ensuring "no or negligible risk", it may only be subject to declaration. (Art. L. 532-3)
Indeed, concerned by the fact that GMOs "may reproduce in the environment and cross national borders, thus affecting other Member States" (Court of Justice of the European Communities), these laws set out in the Environmental Code therefore aim to ensure the safety and control of the use of GMOs without prohibiting it. These laws thus apply to all laboratory studies but also to all industrial production, whether in the field of biofuels, the textile and material industry or the medical field.
Concerning the concrete place of GMOs in France and in Europe, it appears that they tend to be developed, in compliance with the rules mentioned above. As far as France is concerned: "52 [...] Between 2003 and 2006, France is said to have occupied the second position among the Member States, both in terms of the number of applications for authorisation to release for experimental purposes and in terms of the production of GMOs for commercial purposes". Moreover, past events support this point, in particular the example of a placing on the market granted in the Netherlands: In 2007, "Pioneer Overseas Corp. submitted to the competent authority of the Netherlands, [...] an application for authorisation to place on the market food, food ingredients and feed containing genetically modified soybean 305423 [...]". Following an in-depth study, this GMO soybean, "as safe for its intended uses as the conventional counterpart" received a favourable opinion for marketing authorisation (COURT OF FIRST INSTANCE OF THE EUROPEAN UNION, (Seventh Chamber)). This is the very example of a market and legislation that are not set in stone and that aim solely to protect humans and the environment.
GMO acceptance and increasing security of genetic confinement
However, there are still major public concerns about GMOs in Europe. We were able to encounter it directly at the Marburg meet-up. Indeed, at that time, we had not finalized our project and we wanted to use stable genetic constructs to perform phage therapy in order to fight bacterial infections resistant to antibiotics. We were able to hold a workshop, for which we prepared a video presentation (and for which we won the 3rd price! See our video here). We were therefore able to confront our ideas with other iGEMers during this workshop. The students who participated were afraid of GMOs, mainly because the organism is alive and can evolve on its own. We therefore explained to them the principle of interlacing genes and securing genetic information. As the principle was complicated to assimilate, we then popularised interlacing with the image of two genes hugging each other, or like braids. So, the question was whether people would be more confident with interlacing genes. Despite the fact that they were sceptical about the method, more than half of the people felt more able to accept a GMO treatment if it was confined and could not evolve out of control in their bodies.
The same goes with the general public (Court of Justice of the European Communities), and this is why countries are primarily concerned to reassure their citizens and thus facilitate the acceptance of GMOs: "[...] the delays observed are linked in particular [...] to a concern to calm the public order disturbances caused by GMO crops and to facilitate public acceptance of these crops through more ambitious reforms" (Court of Justice of the European Communities).
The study of European legislation therefore tells us that this is a delicate but constantly evolving subject. Our HuGenesS project takes on its full meaning in this legal and social context. It has the dual purpose of reassuring citizens through the very essence of the project but also through our efforts to popularise biology and genetic manipulation, and also to introduce an additional level of safety for the increasingly pronounced use of GMOs in our countries. HuGenesS is becoming a perfectly integrated tool in this world torn between the need for safety and the need to take new paths.
Faced with this observation, we wanted to make sure that our project was in favor of the fundamental values of biocontainment. This is why we created in silico entanglements carried out with the CcdB toxin gene, which could then be used as a kill switch that would be activated in conditions associated with an accidental release in the environment.
Let’s take for example the Passcode Switch. This switch requires the combination of 3 different signals to prevent the production of a toxin. If the organism escapes from this very controlled environment, the toxin will be produced, and the GMO will die. But this genetic circuit contains an Achilles’ heel, ie the possible evolution of the toxin gene. Indeed, if mutations accumulate in the toxin gene or the organism loses it, then the confinement can be bypassed.
But if the toxin gene is entangled with an essential one, a mutation inactivating the toxin could have dire consequences for the gene essential for survival. It is what we want to propose with the entanglement of the CcdB toxin gene. Our solution is then a way to combine stability and security, to offer a better confinement.
Understanding the motivations of the scientists at the genesis of CAMEOS
In spite of the tremendous potential of this method, gene entanglement is not yet widely used by synthetic biologists because of its novelty, the complexity of the mathematical tools used within the software and the need for training to make the software more accessible for biologists.
Indeed, the article (Blazejewski et al, 2019) describing the software CAMEOS is very recent (it was published in August 2019) and totally innovative , but, when we first read it in depth, this article as well as this software remained a “beautiful and magical” mystery. We worked hard to install and get CAMEOS to run smoothly. Despite numerous readings the fundamental mathematical principles of the software were not explained and this slowed down our understanding. But this investigation brought to light many questions about the conception and the origins of the software.
We then came into contact with the authors of the article and had the chance to virtually meet PhD student Thomasz Blazejewski and Dr Harris H. Wang, two of the three co-authors of the article. This extremely enriching discussion provided us with key insights.
One of our first questions was how the authors came up with the idea of creating such software and why they embarked on this adventure. It turned out to be both a real challenge to be able to develop such a software, as well as an answer to the need to secure GEMs. As the entanglement of genes already exists in nature, the idea was to be able to reproduce this phenomenon artificially in order to create new tools for genetic manipulation.
Another question allowed us to understand that the Julia language had been used to make it easier to use Hidden Markov Models (HMMs) as well as Markov Random Fields (MRFs). This made us realise the importance of these mathematical tools, and we felt it was essential to explain them in detail in a modelling of the procedures underlying CAMEOS (Model).
In addition, the authors pointed out that the use of fluorescent proteins in gene entanglement would probably be a problem for us. Indeed, the sensitivity of these proteins is such that if their conformation is slightly modified, their chromophore no longer forms, and then we will have no phenotype. Harris Wang advised us rather towards enzymes or toxins.
Science communication with a broad audience: videos and workshops that make DNA tangible and open the discussion about DNA modifications in GEMs
Thanks to this interview we were more able to understand the whole functioning of CAMEOS. Eager to share the perspectives opened by this technology, we wanted to popularize synthetic biology and the principle of gene entanglement. The goal was to capture the interest of a broad public about genes, the genetic code and how to decode genetic information supported by DNA. Communicating about science with the general public is far from straightforward, yet it is essential because it is this communication that makes it possible to have a global impact on the world, regardless of one’s knowledge in biology.
Our very first public experience with Science Education was the “Apériscience”, a workshop on DNA held at the Jean Cocteau Library in Massy (France) on February 28th, two weeks before France went into lock down caused by the CoViD-19 pandemic. During this evening we welcomed a large public including 3 classes of high school students. The theme of our workshop was DNA: even if our project was not well defined then, this exercise was particularly formative because we were confronted with the incomprehension and reluctance of some young people. The DNA extraction workshop that we set up that evening was a way to make this molecule tangible. These simple and playful manipulations made the public attentive and interested in biology. We were therefore able to go further afterwards, by explaining the principle of synthetic biology and GEMs.
Early March, we got in touch with Mrs. Valérie Lerouyer, who works at the Cité des Sciences, a science museum. Thanks to her interest, we were able to participate in the Fête de la Science weekend in October. For two days, we had the chance to hold a stand on Synthetic Biology and on our HuGenesS project. In the context of our yearlong partnership with other Parisian teams (Partnership), we organised this major event with our partners. Throughout the weekend we were able to introduce the general public to the fundamental concepts of biology with practical workshops on DNA extraction and lots of colourful explanations.
For this week-end, we organised an interactive and fun session during which the public had to decode a sequence of DNA into a protein sequence thanks to the genetic code. The protein sequence was a sentence, and for most people, it was the first time they really understood the principle of the genetic code. Moreover, we had entangled two sequences to decode, so the public could see the idea of multiple messages encoded in different reading phases.
But the public was also made up of science enthusiasts to whom we were able to detail our respective projects and exchange with them about the impact of iGEM on the world. It was a wonderful experience of sharing and popularising science, thanks to which we were able to reach a much wider audience than we had done up until now. We were a source of information and a meeting place for those present, while they were a way for us to improve the visuals of our poster and website, especially with clearer images. Being in contact with other great popularisers and scientists taught us how to present a project and how to capture the public's attention. What are the points to support and those to keep in mind for the most curious? This experience has totally changed the way we integrate our project into the way we present it.
We also tried another type of communication: videos. Thanks to the collaboration of 11 French and Swiss teams, we managed to edit a common video “iGEM FR” telling the story of our teams, projects and regions. This video, which was made public, was also a means of publicising the iGEM competition and the various global issues at stake, on very broad topics that can be tackled and viewed from a new angle. Thanks to the making of this video, we were able to imagine and start the Promotion and Presentation videos requested for the competition. We were able to exchange with the teams on how to capture the video, what made the media attractive and clear or on the contrary soporific, and thus integrate the basic knowledge of the film that some teams had and that our own lacked. This collaboration was essential to our team spirit as well as to the quality of our video production.
This video was the best way to talk about CAMEOS with people from the world, not only iGEMers but everyone interested in the competition or in biology. You can see it in this link. More details in Communication page
Enhancing the adoption of gene entanglement by scientists through communication and tutorials on CAMEOS
But after having popularized the biology and gene entanglement to the large public, we wanted to confront ourselves with the researchers. Indeed, making a technique accessible also means communicating about it and surveying potential stakeholders about their needs and comments. That’s why we decided to interview some researchers in order to know if they had already heard about CAMEOS and what they thought about it.
It turned out that all of the people we contacted had not heard of CAMEOS. Moreover most of them were totally unfamiliar with the very principle of man-made design of sequence entanglement. Indeed, among all our interlocutors, only one person – Anne Lopes – had worked with researchers in bioinformatics interested in the principle of gene tangles, but without knowing CAMEOS.
Dr Gaëlle Lelandais is a researcher in bioinformatics who agreed to take her time to exchange with us. Not knowing iGEM, this competition was a real discovery for her, as was the subject we are working on this year. As a result of reading the article on CAMEOS and our discussions, the principle of genetic confinement imposed itself on her as a real necessity of which she was not fully aware. From then on, she was very interested in our subject, but it remained very vague. We were therefore able to explain our project to her as well as its stakes. As a bioinformatician, she enlightened us on her expectations in software development, gave us her opinion on the strengths of CAMEOS and directed us towards the need for an explanation tutorial. For her, it was absolutely fundamental that we included a concrete and detailed step-by-step example as well as a mathematical explanation of the software's internal functioning. Which we did. Thanks to her vision, we were able to better visualise the expectations of future users in order to adapt the tutorial on the use of CAMEOS adapted to their needs. The tutorial we developed is accessible here.
We then went to see a researcher, who had worked in a team interested in gene tangle and knew the principle. However, she was not familiar with CAMEOS. We had the chance to interview Dr Anne Lopes who studies the emergence of genes de novo from a genomic and structural point of view, in the Biomolecular Informatics Team of Paris-Saclay. Very interested in the evolutionary aspect of gene interlacing, Anne Lopes put her finger on an aspect that we had not considered. She proposed to study whether there are physicochemical similarities between the proteins encoded by the intertwined genes. In particular, this would involve studying hydrophobicity profiles, because in nature many overlapping sequences encoded in different frames present similar profiles (Bartonek et al, 2020). As we did not have the opportunity to do this for our sequences, it seemed obvious to us to include it in the improvements (see further in implementation page) to be proposed.
Similarly, the interview with Dr Matthieu Falque allowed us to continue talking about evolution. He raised the idea of observing the entangled genes existing in nature and trying to artificially entangle them with CAMEOS in order to be able to compare the results. The lack of time due to the health crisis linked to Covid restricted us in our manipulations, and that is why we were not able to test his proposal.
However, together with Anne Lopes, these two researchers interested in evolution opened our project to a new perspectives: the comparison of artificial and natural entanglement in order to better understand the mechanisms and to be able to draw inspiration from them.
Analysis
Finally, the potential of HuGenesS seemed sufficient to extend the research and to push up the use of this technique. So if we want our technique to be considered in the real world, we need to compare it with the various alternatives currently available, establish the technique’s strengths and weaknesses, its potential and the threats it faces. This is why we have carried out a analysis of the actual offer, for a potential commercialization, in order to have a global vision of a technique that could be implemented into the world today.
References
Fang et al, 2017: Fang, Guell, Church & Pfeifer (2017) Heterologous erythromycin production across strain and plasmid construction . Biotechnology Progress . doi:10.1002/btpr.2567
Rosenberget al, 2008: Rosenberg, Oyler, Wilkinson, Betenbaugh (2008) A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution. Current Opinion in Biotechnology , doi.org/10.1016/j.copbio.2008.07.008
Czerwiec et al, 2018: Czerwiec, Idrissitaghki, Imatoukene, Nonus, Brigitte , Nicaud & Rossignol (2018) Optimization of cyclopropane fatty acids production in Yarrowia lipolytica . Yeast . doi: 10.1002/yea.3379
Torres et al, 2016: Leticia Torres, Antje Krüger, Eszter Csibra, Edoardo Gianni, Vitor B Pinheiro, Synthetic biology approaches to biological containment, pre-emptively tackling potential risks, Essays in Biochemistry (2016) 60 393–410, DOI: 10.1042/EBC20160013
Simon et al, 20016: Anna J. Simon, Andrew D. Ellington, Recent advances in synthetic biosafety, 31 Aug 2016, 5(F1000 Faculty Rev):2118 , https://doi.org/10.12688/f1000research.8365.1
Court of Justice of the European Communities / 9 December 2008 / n° C-121/07 https://www.dalloz.fr/documentation/Document?id=CJCE_LIEUVIDE_2008-12-09_C12107#_
COURT OF FIRST INSTANCE OF THE EUROPEAN UNION, (Seventh Chamber) / Judgment of 14 March 2018 / In Case T-33/16 https://www.dalloz.fr/documentation/Document?id=A249675
Bartonek et al, 2020: Lukas Bartonek, Daniel Braun, and Bojan Zagrovic, Frameshifting preserves key physicochemical properties of proteins, PNAS March 17, 2020 117 (11) 5907-5912; first published March 3, 2020; https://doi.org/10.1073/pnas.1911203117
Blazejewski et al, 2019: Tomasz Blazejewski, Hsing-I Ho and Harris H. Wang, Synthetic sequence entanglement augments stability and containment of genetic information, Science 365 (6453), 595-598, DOI: 10.1126/science.aav5477