Team:Toulouse INSA-UPS/Contribution
Contribution
Making a useful contribution for future iGEM Teams has been our leitmotiv since the start of our project. We are definitely convinced that the experience accumulated by each iGEM Teams is of outstanding interest for the future iGEMers. We profited a lot of what our predecessors have transcribed in their wiki and we have decided to do our best to be as useful as possible to the next generations of iGEM students.
A new prospect field for iGEM
From the first brainstorming session, we tried to think out of the box to identify a new and innovative project. We felt that even after around 3000 projects, it should be possible to find new applications to synthetic biology. So, our starting point was to engage in an innovative theme opening up multiple possibilities for future iGEM topics. If the perfume industry knows about biotechnology, the use of synthetic biology in this field is only in its infancy. This means there are an incredible number of exciting possibilities, of fragrances to be produced at lower costs, of rare scents to sustainably produce, and even new never smelled before molecules to be created!
Figure 1: “L’orgue à parfum”, the perfumer’s organ is a professional piece of furniture designed to store most of the bottles of raw materials used by perfumers.
We were very surprised to find hardly any project about the use of synthetic biology
to produce fragrances for the perfume industry (but for iGEM Kent 2014).
Consequently, we feel that our most important contribution for future iGEM teams is
the opening of this field.
We foresee our wiki to be scrutated in the next future. We designed our wiki pages
to contain useful tips and information to help designing and experimenting new
biosynthetic fragrances. The next paragraphs will describe our main contributions
that can be found on the pages of this wiki.
New documentation to existing Parts
Figure 2: iGEM Registry.
The project developed this year by the iGEM team of Toulouse has created many parts for metabolic engineering. Others parts we used were already present in the iGEM registry. Here are some we used and for whose we gathered new information:
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BBA_K1969005: this part corresponds to the pCUP1 promoter, inducible by the addition of copper in the medium. This part had not been characterized before, but the team showed this year that it is functional for expression in S. cerevisiae.
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BBa_K2117000 and BBa_K2117005: those parts correspond to the pTEF1 promoter. They had been characterized before for expression in Y. lipolitica, but the iGEM Toulouse 2021 team showed that it is also functional for expression in S. cerevisiae.
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BBa_K3629011: this part corresponds to the nsrR resistance gene, useful for transformants selection. This part had not been characterized before and its expression was supposed to be optimized for the chassis Y. lipolitica. We demonstrated here that this part, once codon optimized for S. cerevisiae, is also functional in this chassis.
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BBa_K1323012: this part corresponds to the spcR resistance gene and had not been characterized before. We showed this year that it is functional for expression in the cyanobacterium S. elongatus.
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BBa_K1313004: this part corresponds to the neoR resistance gene, useful for E. coli transformants selection with neomycin. We demonstrated here that this part is also functional in S. cerevisiae for selection on G418.
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BBa_K1969004: this part corresponds to the Gal1 promoter, lacking the kozak sequence. No experience results were obtained for it. We demonstrated here this version of the Gal1 promoter is functional in S. cerevisiae.
Strains and new parts for a fragrance producer yeast chassis
Many fragrances are issued from the terpene pathway. Here we showed that using a yeast strain modified to produce lycopene is a good chassis to produce a broad range of interesting fragrances that are relevant for industry. We had to engineer this strain further to remove a resistance marker. We then proved that this strain can accept three constructions at three new integration sites never used in iGEM. We also showed that it can be used successfully to produce ionones, and even other derivative molecules like dihydro-ionone. All these strains will be made available to future teams. As for parts, all the information to use our integration sites, our terpene-related enzymes, and a lot more are present on our Parts page and the registry.
Figure 3: Native and heterologous pathways for the production of violet. α-ionone, β-ionone, dihydro-β-ionone, and linalool terpenes in Saccharomyces cerevisiae.
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Integrative site in locus XII-1 of S. cerevisiae genome:
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Integrative up site in locus X-3 of S. cerevisiae genome:
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Integrative up site in locus XII-4 of S. cerevisiae genome:
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Linalool and dihidro-β-ionone induction system and expression in S. cerevisiae genome:
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β-ionone induction systems and expression in S. cerevisiae (enzymatic fusion)
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α-ionone induction systems and expression in S. cerevisiae (enzymatic fusion)
A Phototroph Guide
Figure 4: Picture of our cyanobacteria bioreactor experiment.
While extremely promising for the development of sustainable processes, working with
cyanobacteria is not a piece of cake. Other teams have made the same observation and
we
decided to join forces to gather our observations, successes and failures. From
these was
issued a Prototroph Guide to help the future iGEMers to
avoid
making the same mistakes and improve the chances of success of their projects.
At our team level, we successfully modified the cyanobacterium genome through
triparental
conjugation using a not before described integration site. This information could be
found
in our Protocol
page and on the registry:
Here is our guide for future iGEM teams to use phototrophic organisms:
Other experimental tips
We were lucky enough to succeed in our cloning, integration and our most critical analytical experiments. Anyway, this has not been a straight and easy road and we committed some errors too. Since those could be of interest for future teams to avoid reproducing them, we added in our Results section tips and troubleshooting advice in green.
Figure 5: Cryostocks of our final strains.
Modeling microbial consortia
We developed a dynamic and predictive coculture model, which allowed us to demonstrate the feasibility of our project, optimize it and dimension a pilot production unit. This modeling approach is fully modular and has been designed to be reused by any team interested in modeling dynamic synthetic consortia. It is made available in the form of a Jupyter Notebook that can be downloaded here. Overall, our strategy demonstrates how modeling can be used as a central tool to understand and optimize microbial communities for biotechnological applications, as well as to support the implementation of an industrial biotechnology project. We are convinced that the proposed approach - and its associated open-source implementation - will serve future iGEM teams participating in the Manufacturing track.
Figure 6: Summary of modeling success.
Exemplifying our IHP as a roadmap to success
Our project succeeded only because of many feedback we had from specialists and non-specialists. From this positive experience, we were able to highlight key points for a successful IHP. For instance, it appears very clear to us that a manufacturing effort (our track) has to be grounded on the real life and real situation, in most cases, with a true company interested in the iGEM team effort. Another key point for us is to carefully define all the steps of the project and to find appropriate external persons to discuss each of these steps (company, scientist, general public, etc depending on the step). All these considerations have been summed up on our IHP page.
Figure 7: Our IHP effort.
A detailed entrepreneurship approach to dimension a plant and its CO2, energy and cost balances
Figure 8: Flowsheet of the industrial installation.
As we are competing in the Manufacturing track, our entrepreneurship effort had to go a step further. Above classic SWOT or business risk analysis, we engaged in a whole dimensioning of what could be our future plant (see Supporting Entrepreneurship part). Our aim was to be able to predict how our manufacturing solution could perform not only in term of economic viability but also in term of CO2 balances and energy consumption. This is a tedious job to do, especially to gather information, but in this context of global warming and ecological transition, it has to be done. We think this will be of an upmost interest to any team in the same field of research, or simply willing to anticipate the consequence of their project.
Open source board game
Figure 9: Our board game.
Finally, we designed a game to explain synthetic biology and perfume notions. While this game is specific to our topic, we realized that a board game conception is not so easy and spent a lot of time designing the elements (board, cards, game pawns). This is why we have decided to but all the specifications of the elements and the detailed way to produce them on our wiki, so that they can be easily downloaded and modified by any team willing to engage in the production of a board game.
During the development of our game we did some research to understand how we could adapt it for visually impaired people. All the information we gathered is available for other teams to use.
