Team:Sorbonne U Paris/Poster
Poster: Sorbonne_U_Paris
THE CHLAMY CLEANER: A microalgae filter to purify water
Authors:
Antonin Chevenier, Gabriel Chemin, Lorenzo Colombo, Pierre Foucault, Charlotte Joly, Juliette Mandelbrojt, Chloé Piriou, Alexandra Teyssou, Béatrice Urbah - Members of the iGEM Sorbonne Team from Sorbonne Université.
Introduction
For the 2024 Olympic Games, Paris wants to host the triathlon swimming events in the Seine. However, the water is polluted: pesticides, hormones and antibiotics are present and might have negative effects on the environment and on human health. Our goal is to develop a solution to purify water. Using Chlamydomonas reinhardtii as a chassis, we designed a microalgae filter capable of retaining and degrading these harmful compounds. We focused our efforts on Atrazine, a banned herbicide still detectable in the Seine. We expressed three enzymes from the bacteria genera Pseudomonas in C. reinhardtii using the Golden Gate Modular Cloning (MoClo). This newly added degradation pathway aims at degrading Atrazine into a less hazardous product. To ensure additional safety, we integrated a "kill switch" device based on UV-sensitive nuclease genetic circuit leading to the death of any microalgae escaping into the wild.
Inspiration
The Seine Basin covers a total area of more than 78,000 km², while the Seine and its tributaries flow through areas of high agricultural and industrial activity and high population density. On one hand, the runoff water is loaded with contaminants of various origins before reaching the river circulation. On the other hand, wastewater from the use of running water is highly loaded with pollutants. Nowadays, it is not directly discharged into waterways. Wastewater treatment plants can treat a large part of the pollution generated. However, in spite of all their advantages, these plants are not efficient enough to treat certain categories of micropollutants, including antibiotics, synthetic hormones and pesticides.
The problem of river water pollution is complex and multifactorial, which is why improving water treatment is a major environmental issue but also a public health issue. It is in this context that we felt it was necessary to work on the degradation of these organic pollutants.
The problem of river water pollution is complex and multifactorial, which is why improving water treatment is a major environmental issue but also a public health issue. It is in this context that we felt it was necessary to work on the degradation of these organic pollutants.
Problem
Atrazine is a herbicide that acts by blocking plant photosynthesis. It belongs to the products of organic synthesis: triazines. It was used mainly as a weed killer for corn fields for forty years, from its introduction in 1960 until its prohibition, decided in 2001. Its toxicity is proven on the aquatic environment and for humans (Oka T et al. 2008; Ralston-Hooper K et al. 2009; Sánchez OF et al. 2020; Jablonowski ND et al. 2011) and atrazine is classified as a "harmful product". However, despite of being banned, it is still detectable in the water of the Seine.
Idea
We expressed four enzymes from the bacteria genera Pseudomonas in C. reinhardtii using the Golden Gate Modular Cloning (MoClo). This newly added degradation pathway aims at degrading Atrazine into a less hazardous product. In order not to further impact the environment, we integrated a "kill switch" device based on UV-sensitive nuclease genetic circuit leading to the death of any microalgae escaping into the wild.
MoClo
The MoClo relies on the Golden Gate cloning strategy (Weber et al. 2011) which allows a directional assembly of multiple DNA fragments into a one-pot, one-step reaction, thanks to type IIS restriction enzymes. Each type of basic parts has a position defined by the fusion sites on both sides (see below).
(Crozet et al. 2018)
For Chlamydomonas reinhardtii, a specific MoClo toolkit has been developed (Crozet et al. 2018), and we used their standard for the construction of our parts.
There are three Levels of MoClo Parts (shown below):
Level 0: Basic part (ex: promoters, CDS, 5’UTR, tags etc.) which contains different fusion sites depending on their position in the transcriptional unit.
Level 1: Transcriptional unit where up to 10 level 0 parts can be assembled.
Level M: Composite multigenic part consisting of assembly up to 7 level 1 parts.
A multigenic level M plasmide was designed for each of our 2 main functions: atrazine degradation and the kill-switch device.
(Crozet et al. 2018)
For Chlamydomonas reinhardtii, a specific MoClo toolkit has been developed (Crozet et al. 2018), and we used their standard for the construction of our parts.
There are three Levels of MoClo Parts (shown below):
Level 0: Basic part (ex: promoters, CDS, 5’UTR, tags etc.) which contains different fusion sites depending on their position in the transcriptional unit.
Level 1: Transcriptional unit where up to 10 level 0 parts can be assembled.
Level M: Composite multigenic part consisting of assembly up to 7 level 1 parts.
A multigenic level M plasmide was designed for each of our 2 main functions: atrazine degradation and the kill-switch device.
Atrazine degradation
We imagined an engineered microalgae able to express the atrazine degradation pathway. This new function is the heart of our concept: making Chlamydomonas reinhardtii the ultimate tool for water depollution by enhancing its bioremediation spectrum. We selected 3 genes: atzA, atzB and atzC.
These 3 genes encode enzymes which metabolize atrazine into cyanuric acid, a less toxic compound.
The first gene in the degradation pathway, atzA, encodes for atrazine chlorohydrolase which catalyzes atrazine dechlorination to hydroxyatrazine. It was first identified in 1996 (de Souza et al. 1996).
The second gene (atzB) encodes for hydroxyde-chloro-atrazine ethylaminohydrolase (atzB) and catalyzes the hydroxyatrazine deamidation, yielding N-isopropylammelide (Govantes et al. 2010).
Finally, N-isopropylammelide isopropyl amidohydrolase (atzC) transforms N-isopropylammelide to cyanuric acid and isopropylamine. (Sadowsky et al. 1998).
These enzymes are well described in the litterature and known since the 90s. They were isolated and characterized from the Pseudomonas sp. ADP strain, the most studied bacteria able to use atrazine as a carbon source (de Souza et al. 1996; de Souza et al. 1998).
These 3 genes encode enzymes which metabolize atrazine into cyanuric acid, a less toxic compound.
The first gene in the degradation pathway, atzA, encodes for atrazine chlorohydrolase which catalyzes atrazine dechlorination to hydroxyatrazine. It was first identified in 1996 (de Souza et al. 1996).
The second gene (atzB) encodes for hydroxyde-chloro-atrazine ethylaminohydrolase (atzB) and catalyzes the hydroxyatrazine deamidation, yielding N-isopropylammelide (Govantes et al. 2010).
Finally, N-isopropylammelide isopropyl amidohydrolase (atzC) transforms N-isopropylammelide to cyanuric acid and isopropylamine. (Sadowsky et al. 1998).
These enzymes are well described in the litterature and known since the 90s. They were isolated and characterized from the Pseudomonas sp. ADP strain, the most studied bacteria able to use atrazine as a carbon source (de Souza et al. 1996; de Souza et al. 1998).
Kill-switch
For our project, developing a kill-switch is essential, as our modified algae is supposed to be in contact with the Seine’s water. The algae has to be contained in filters in order to prevent the spread of our organisms in the river in an uncontrolled way. However, these filters may not be completely infallible. For additional safety, we designed an ingenious kill switch system, causing the algae to die when it manages to pass through the filter. This system is based on UV-light. The algae will be cultivated and used under so-called ‘high-pass’ color filters that prevent UV light from passing through. Therefore, if the algae goes out of this chromatic filter, it dies prematurely because of the exposition to the light spectrum.
To do so, we have chosen the micrococcal nuclease from Staphylococcus aureus(Heins et al, 1967) to fragment genomic DNA and induce programmed cell death. The nuclease is coupled with a nuclease localization sequence, a transmembrane domain and a TEV recognition/cleavage site in between.
In parallel, we designed two fusion proteins, each one composed of the C-terminal or the N-terminal splitted part of the TEV protease, each coupled with UVR-8 or COP1. These latter are known to hetero-dimerize after UV-light exposure (Cloix et al, 2012).
As long as the two fusion proteins are separated, the protease cannot be active. When the fusion proteins dimerize, the TEV protease is reconstituted, resulting in a proteolytic activity for a specific TEV recognition site (Michael C Wehr et al, 2006).
The reconstituted TEV protease liberates the micrococcal nuclease from its membrane anchor by cleaving the TEV site and separating the nuclease and the transmembrane domain. Then, the nuclease will be able to migrate in the nucleus and fragment the genomic DNA.
To do so, we have chosen the micrococcal nuclease from Staphylococcus aureus(Heins et al, 1967) to fragment genomic DNA and induce programmed cell death. The nuclease is coupled with a nuclease localization sequence, a transmembrane domain and a TEV recognition/cleavage site in between.
In parallel, we designed two fusion proteins, each one composed of the C-terminal or the N-terminal splitted part of the TEV protease, each coupled with UVR-8 or COP1. These latter are known to hetero-dimerize after UV-light exposure (Cloix et al, 2012).
As long as the two fusion proteins are separated, the protease cannot be active. When the fusion proteins dimerize, the TEV protease is reconstituted, resulting in a proteolytic activity for a specific TEV recognition site (Michael C Wehr et al, 2006).
The reconstituted TEV protease liberates the micrococcal nuclease from its membrane anchor by cleaving the TEV site and separating the nuclease and the transmembrane domain. Then, the nuclease will be able to migrate in the nucleus and fragment the genomic DNA.
Results
Chemical analysis of Seine water
Analysis of four samples from four different locations around the Seine bassin:
The samples were analysed in the METIS laboratory (Organic contaminant departement) where they performed a chemical analysis by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Here is the list of the pollutants detected:
Atrazine and two of its degradation products, DEA (diethyl-atrazine) and DIA (desisopropyl-atrazine), were included in the 17 different pollutants detected (LOQ = limits of quantification).
Parts cloning
Here is an overview of our progress in the cloning of our parts:
Unfortunately, due to new covid-related safety norms, we were not able to transform our Chlamydomonas reinhardtii strain with our level M plasmid.
Atrazine and cyanuric acid toxicity assays on Chlamydomonas reinhardtii:
Two types of toxicity assays were performed:
We tested a range of pollutant concentration from 0 to 1500 µg/L.
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Plate assays results:
- Atrazine alters the growth of Chlamydomonas reinhardtii strain D66
- Toxicity threshold: 250µg/L in TAP medium
- Cyanuric acid did alter the growth of Chlamydomonas reinhardtii strain D66 in TAP medium
Algem® photobioreactor assays results:
We tested a range of concentration from 0 to 500 µg/L:
Growth curve of Chlamydomonas reinhardtii D66 strain D66 at various atrazine concentrations: Green : 0 µg/L ; yellow : 77 µg/L ; orange : 250 µg/L ; red : 500 µg/L.
- All the atrazine condition hampers the microalgae growth
- Toxicity threshold at 77µg/L in TAP medium
A lower toxicity threshold was expected since the Algem has more precise data.
Future work:
Unfortunately, due to the new covid-related safety norms which cut our lab access, we were not able to finish our project. Here is a list of tasks that would have to be completed going forward:
- Finish the cloning of the missing parts of the kill-switch
- Transform our microalgae with the Level M plasmid
- Characterize our parts expression level (detection of HA-tag)
- Test the efficiency of the kill-switch device (Cell death under UV-light)
- Test the efficiency of the atrazine degradation pathway: toxicity assays with the engineered microalgae, lower toxicity threshold expected
- Optimized the toxicity assays by using HSM (minimum medium) or sterilized Seine water as media.
Analysis of four samples from four different locations around the Seine bassin:
The samples were analysed in the METIS laboratory (Organic contaminant departement) where they performed a chemical analysis by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Here is the list of the pollutants detected:
Atrazine and two of its degradation products, DEA (diethyl-atrazine) and DIA (desisopropyl-atrazine), were included in the 17 different pollutants detected (LOQ = limits of quantification).
Parts cloning
Here is an overview of our progress in the cloning of our parts:
Unfortunately, due to new covid-related safety norms, we were not able to transform our Chlamydomonas reinhardtii strain with our level M plasmid.
Atrazine and cyanuric acid toxicity assays on Chlamydomonas reinhardtii:
Two types of toxicity assays were performed:
We tested a range of pollutant concentration from 0 to 1500 µg/L.
Plate assays results:
- Atrazine alters the growth of Chlamydomonas reinhardtii strain D66
- Toxicity threshold: 250µg/L in TAP medium
- Cyanuric acid did alter the growth of Chlamydomonas reinhardtii strain D66 in TAP medium
Algem® photobioreactor assays results:
We tested a range of concentration from 0 to 500 µg/L:
- All the atrazine condition hampers the microalgae growth
- Toxicity threshold at 77µg/L in TAP medium
A lower toxicity threshold was expected since the Algem has more precise data.
Future work:
Unfortunately, due to the new covid-related safety norms which cut our lab access, we were not able to finish our project. Here is a list of tasks that would have to be completed going forward:
- Finish the cloning of the missing parts of the kill-switch
- Transform our microalgae with the Level M plasmid
- Characterize our parts expression level (detection of HA-tag)
- Test the efficiency of the kill-switch device (Cell death under UV-light)
- Test the efficiency of the atrazine degradation pathway: toxicity assays with the engineered microalgae, lower toxicity threshold expected
- Optimized the toxicity assays by using HSM (minimum medium) or sterilized Seine water as media.
Contribution
IGEM is based on team achievement and contributing to a commun registry to help future projects. Thus, we registered 11 basic parts and 2 composite ones (see below) and completed the basic part RBC2i1 (BBa_K2703000) page with bibliographic research about introns and their potential as a tool for synthetic biology.
Here is the list of the submitted parts:
Introns, non-coding sequences found in eukaryotic genes, can elicit an increased level of mRNA expression of nuclear transgenes in eukaryotic organisms. This process is called Introns-mediated Enhancement (IME). Understanding this process remains a hot topic issue and the answer would have a tremendous impact in synthetic biology.
RBC2i1 (BBa_K2703000) is the first intron of the second small subunit of the Rubisco enzyme gene (RBSC2). It is well described and reviewed in the litterature and is often used to study the IME mechanism because of the high transcription levels of the RBSC2 gene. The microalgae Chlamydomonas reinhardtii is a suitable model organism to study this process due to the presence of the RBCS2 gene and the high intron richness of its genome (Merchant et al. 2017).
Here is an overview of the findings we implemented:
- RBCS2i1 is frequently used with the PsaD (BBa_K1547005) (Baier et al. 2020; Baier et al. 2018; Schroda et al. 2019) promotor and has been studied with various other (Melissa et al. 2016)
- Transcript abundance can be enhanced up to 5,5 times in Chlamydomonas reinhardtii (Baier et al. 2018; Jaeger et al. 2019)
- The intron must be placed in the 5’UTR region for maximal enhancement (still elicit an increase up to 3,7 times if inserted in the CDS) (Baier et al. 2020)
- RSC2i1 can be repeated up to 3 times or coupled with RBCSi2 to reach maximal enhancement (Baier et al. 2020)
- RSC2i1 truncated (from 145 to 115pb ) sequence can maintain the same IME effect (Baier et al. 2020)
- Greater truncation (70pb) still improves the transgene expression by 3,8 times (Baier et al. 2020)
Jaeger and his team created a software to facilitate intron-enriched DNA sequence design, compatible with MoClo’s level 0 parts : Intronserter (Jaeger et al. 2019).
Here is the list of the submitted parts:
Introns, non-coding sequences found in eukaryotic genes, can elicit an increased level of mRNA expression of nuclear transgenes in eukaryotic organisms. This process is called Introns-mediated Enhancement (IME). Understanding this process remains a hot topic issue and the answer would have a tremendous impact in synthetic biology.
RBC2i1 (BBa_K2703000) is the first intron of the second small subunit of the Rubisco enzyme gene (RBSC2). It is well described and reviewed in the litterature and is often used to study the IME mechanism because of the high transcription levels of the RBSC2 gene. The microalgae Chlamydomonas reinhardtii is a suitable model organism to study this process due to the presence of the RBCS2 gene and the high intron richness of its genome (Merchant et al. 2017).
Here is an overview of the findings we implemented:
- RBCS2i1 is frequently used with the PsaD (BBa_K1547005) (Baier et al. 2020; Baier et al. 2018; Schroda et al. 2019) promotor and has been studied with various other (Melissa et al. 2016)
- Transcript abundance can be enhanced up to 5,5 times in Chlamydomonas reinhardtii (Baier et al. 2018; Jaeger et al. 2019)
- The intron must be placed in the 5’UTR region for maximal enhancement (still elicit an increase up to 3,7 times if inserted in the CDS) (Baier et al. 2020)
- RSC2i1 can be repeated up to 3 times or coupled with RBCSi2 to reach maximal enhancement (Baier et al. 2020)
- RSC2i1 truncated (from 145 to 115pb ) sequence can maintain the same IME effect (Baier et al. 2020)
- Greater truncation (70pb) still improves the transgene expression by 3,8 times (Baier et al. 2020)
Jaeger and his team created a software to facilitate intron-enriched DNA sequence design, compatible with MoClo’s level 0 parts : Intronserter (Jaeger et al. 2019).
Integrated human practices
Sing-Pei Yu’s interview:
As our project targets the problem of water pollution, we wanted to broaden our knowledge by focusing on one type of pollutant: microplastics. Therefore we interviewed Sing-Pei Yu, a researcher working on microplastics effects on marine wildlife, with barnacles as an animal model. Her studies show that microplastics cause higher mortality on barnacles culture generation F1. More research is needed to support this hypothesis but it shows a clear impact of microplastics on aquatic wildlife, thus it could have an impact on the deregulation of the trophic network. Finally, this interview helped us understand the wild range of water pollutants, the environmental impact of water pollution and the importance of raising public awareness about it. Aside from the scientific aspects discussed she also gave us a lot of advice on how to carry out our project.
Pi Collén and Pierre Rocheteau’s interviews:
Pi Collén and Pierre Rocheteau work for the Olmix group, a company specialized in algae valorization. This enabled us to visualize the different possible applications for the use of algae in industry. In fact, it was very interesting as the use of their products in animal husbandry participates in reducing the use of antibiotics, thus decreasing their release in fields and crops and finally in the water: the aim of our project. We also discussed the implementation of our project and more specifically our kill switch system to prevent the escape of our modified algae in the environment. With their pertinent remarks, we have also considered a more diverse system with different microbial types. This would ensure a complete degradation of the targeted pollutants and could make our system more reliable because monocultures are more sensitive than an entire ecosystem.
Julien Chabrol’s interview:
Julien Charbol is an expert in instrumentation at Veolia with an expertise in water treatment in purification stations. Wishing to implement our system within wastewater treatment plants, we needed to understand the various challenges we were going to face. While discussing with him, he helped us think about another possible implementation which would be to use our system to treat industrial effluents. Indeed, the government increasingly encourages industries to equip themselves with devices of this type. Manufacturers would therefore represent a second user to whom we could implement our project.
As our project targets the problem of water pollution, we wanted to broaden our knowledge by focusing on one type of pollutant: microplastics. Therefore we interviewed Sing-Pei Yu, a researcher working on microplastics effects on marine wildlife, with barnacles as an animal model. Her studies show that microplastics cause higher mortality on barnacles culture generation F1. More research is needed to support this hypothesis but it shows a clear impact of microplastics on aquatic wildlife, thus it could have an impact on the deregulation of the trophic network. Finally, this interview helped us understand the wild range of water pollutants, the environmental impact of water pollution and the importance of raising public awareness about it. Aside from the scientific aspects discussed she also gave us a lot of advice on how to carry out our project.
Pi Collén and Pierre Rocheteau’s interviews:
Pi Collén and Pierre Rocheteau work for the Olmix group, a company specialized in algae valorization. This enabled us to visualize the different possible applications for the use of algae in industry. In fact, it was very interesting as the use of their products in animal husbandry participates in reducing the use of antibiotics, thus decreasing their release in fields and crops and finally in the water: the aim of our project. We also discussed the implementation of our project and more specifically our kill switch system to prevent the escape of our modified algae in the environment. With their pertinent remarks, we have also considered a more diverse system with different microbial types. This would ensure a complete degradation of the targeted pollutants and could make our system more reliable because monocultures are more sensitive than an entire ecosystem.
Julien Chabrol’s interview:
Julien Charbol is an expert in instrumentation at Veolia with an expertise in water treatment in purification stations. Wishing to implement our system within wastewater treatment plants, we needed to understand the various challenges we were going to face. While discussing with him, he helped us think about another possible implementation which would be to use our system to treat industrial effluents. Indeed, the government increasingly encourages industries to equip themselves with devices of this type. Manufacturers would therefore represent a second user to whom we could implement our project.
Science and communication - Articles, comic book & social networks
Articles:
We wrote several articles for a student scientific journal of our university called "I science therefore I am", about our project, GMOs and pesticides. We also participated in the Maastricht’s Proceedings Journal collaboration for which we wrote an overview of the successes and disasters of genetic engineering. This was a cooperative work of peer reviewing other teams' articles and ours was reviewed by the teams MI and Manipal BioMachines. The aim was to ultimately develop a printed version of the best papers which were voted by the participating teams and ours got selected!
Escape Game “Seine est Sauve”:
For the Science Festival hosted by Sorbonne Université, we created an online escape game called "Seine est Sauve" (Seine is Safe) where the aim was to help young scientists give superpowers to an algae in order to cleanse Seine’s water. We based the game development on a comic book we created which is described below. Throughout this game, we explained to children what DNA is and the complementarity of the bases as well as gave them an introduction to microscope usage.
Comic Book “Seine est Sauve”:
This comic book presents our project in a fun way for children (and adults too): young scientists give superpowers to an algae called ChlamChlam (Chlamydomonas reinhardtii) in order to help her clean Seine’s water. With this comic book, we give an introduction of biological terms to children. It was created with the help of a graphic designer, Floki.
Social Media:
Even if we were planning on being very present on our different social medias, the lockdown encouraged us to innovate and adapt our communication even more with these tools. We developed our social media by offering regular infographics on our project, as well as a series of short videos "1 day, 1 activity" during several days at the start of the first lockdown which was in March in France. This was an opportunity to present each member of our team in an original way while entertaining our followers during this very special period and propose safe and fun activities to do inside our homes. We also ran a quiz on our Instagram. The aim was to present iGEM, our project as well as synthetic biology and make our audience participate. Many people engaged in this quiz and we had a lot of positive feedback which was very encouraging for us. We also promoted the collaborations we have made with the different iGEM teams.
We wrote several articles for a student scientific journal of our university called "I science therefore I am", about our project, GMOs and pesticides. We also participated in the Maastricht’s Proceedings Journal collaboration for which we wrote an overview of the successes and disasters of genetic engineering. This was a cooperative work of peer reviewing other teams' articles and ours was reviewed by the teams MI and Manipal BioMachines. The aim was to ultimately develop a printed version of the best papers which were voted by the participating teams and ours got selected!
Escape Game “Seine est Sauve”:
For the Science Festival hosted by Sorbonne Université, we created an online escape game called "Seine est Sauve" (Seine is Safe) where the aim was to help young scientists give superpowers to an algae in order to cleanse Seine’s water. We based the game development on a comic book we created which is described below. Throughout this game, we explained to children what DNA is and the complementarity of the bases as well as gave them an introduction to microscope usage.
Comic Book “Seine est Sauve”:
This comic book presents our project in a fun way for children (and adults too): young scientists give superpowers to an algae called ChlamChlam (Chlamydomonas reinhardtii) in order to help her clean Seine’s water. With this comic book, we give an introduction of biological terms to children. It was created with the help of a graphic designer, Floki.
Social Media:
Even if we were planning on being very present on our different social medias, the lockdown encouraged us to innovate and adapt our communication even more with these tools. We developed our social media by offering regular infographics on our project, as well as a series of short videos "1 day, 1 activity" during several days at the start of the first lockdown which was in March in France. This was an opportunity to present each member of our team in an original way while entertaining our followers during this very special period and propose safe and fun activities to do inside our homes. We also ran a quiz on our Instagram. The aim was to present iGEM, our project as well as synthetic biology and make our audience participate. Many people engaged in this quiz and we had a lot of positive feedback which was very encouraging for us. We also promoted the collaborations we have made with the different iGEM teams.
Science and communication - Workshops & events
Quiz of the Science Festival:
Each year, Sorbonne Université hosts the Science Festival bringing together families and students from primary schools, middle schools, high schools and college students as well as teachers and researchers. This year, the event was offered in digital form. We chose to run a quiz with questions related to iGEM, synthetic biology and water pollution. It was broadcasted live on the Sorbonne Université Youtube channel.
Cité des sciences:
The Cité des Sciences et de l’Industrie (Science and Industry museum) in Paris was also taking part in this national Science Festival. During a week-end, we hosted a booth there with all the other parisians teams. We presented our respective projects and two different workshops. For the first one, we used a microscope connected to a projector allowing us to explain the concept of the particles invisible to the naked eye. The second workshop was based on the extraction of DNA of a kiwi to explain DNA is. The participants were very diverse, each presentation was different and very enriching for both parts.
Maison de la Nature:
We had the chance to carry out a second workshop in the “Maison de la Nature et de l’Arbre” (House of nature and trees) in Meudon. We offered a brief history of wastewater treatment and its function, and we developed its mechanism. Then the participants tried to purify ‘dirty’ water using different homemade filters. Lastly, we offered a microscope presentation, during which we taught the use of a microscope to each participant and offered them different plates to observe, such as our beloved chassis, Chlamydomonas reinhardtii.
iGEM Worldwide Virtual Meetup hosted by Parisian teams:
We hosted The iGEM Worldwide Virtual Meetup with all the parisian teams (iGEM Ionis, iGEM Go Paris Saclay, iGEM Paris Bettencourt and iGEM Evry). During this iGEMeetParis week-end, there were inspiring guest talks, workshops, team project presentations, a Bio-Entrepreneurship Hackathon held by two After iGEM Ambassadors, Nana Oye Djan and Ojas Tulsyan, and of course, fun social activities to end the day.
Each year, Sorbonne Université hosts the Science Festival bringing together families and students from primary schools, middle schools, high schools and college students as well as teachers and researchers. This year, the event was offered in digital form. We chose to run a quiz with questions related to iGEM, synthetic biology and water pollution. It was broadcasted live on the Sorbonne Université Youtube channel.
Cité des sciences:
The Cité des Sciences et de l’Industrie (Science and Industry museum) in Paris was also taking part in this national Science Festival. During a week-end, we hosted a booth there with all the other parisians teams. We presented our respective projects and two different workshops. For the first one, we used a microscope connected to a projector allowing us to explain the concept of the particles invisible to the naked eye. The second workshop was based on the extraction of DNA of a kiwi to explain DNA is. The participants were very diverse, each presentation was different and very enriching for both parts.
Maison de la Nature:
We had the chance to carry out a second workshop in the “Maison de la Nature et de l’Arbre” (House of nature and trees) in Meudon. We offered a brief history of wastewater treatment and its function, and we developed its mechanism. Then the participants tried to purify ‘dirty’ water using different homemade filters. Lastly, we offered a microscope presentation, during which we taught the use of a microscope to each participant and offered them different plates to observe, such as our beloved chassis, Chlamydomonas reinhardtii.
iGEM Worldwide Virtual Meetup hosted by Parisian teams:
We hosted The iGEM Worldwide Virtual Meetup with all the parisian teams (iGEM Ionis, iGEM Go Paris Saclay, iGEM Paris Bettencourt and iGEM Evry). During this iGEMeetParis week-end, there were inspiring guest talks, workshops, team project presentations, a Bio-Entrepreneurship Hackathon held by two After iGEM Ambassadors, Nana Oye Djan and Ojas Tulsyan, and of course, fun social activities to end the day.
References
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2. Baier T, Wichmann J, Kruse O, Lauersen KJ. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Res. (2018).
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6. Daniel Jaeger, Thomas Baier, Kyle J. Lauersen. Intronserter, an advanced online tool for design of intron containing transgenes. Algal Research. (2019).
7. Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. (2008).
8. Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S. A golden gate modular cloning toolbox for plants. ACS Synth Biol. (2014).
9. Govantes F, García-González V, Porrúa O, Platero AI, Jiménez-Fernández A, Santero E. Regulation of the atrazine-degradative genes in Pseudomonas sp. strain ADP. FEMS Microbiol Lett. (2010).
10. Heins JN, Suriano JR, Taniuchi H, Anfinsen CB. Characterization of a nuclease produced by Staphylococcus aureus. J Biol Chem. (1967).
11. Hom-Diaz A, Llorca M, Rodríguez-Mozaz S, Vicent T, Barceló D, Blánquez P. Microalgae cultivation on wastewater digestate: β-estradiol and 17α-ethynylestradiol degradation and transformation products identification. J Environ Manage. (2015).
12. Jablonowski ND, Schäffer A, Burauel P. Still present after all these years: persistence plus potential toxicity raise questions about the use of atrazine. Environ Sci Pollut Res Int. (2011).
13. Klewer L, Wu YW. Light-Induced Dimerization Approaches to Control Cellular Processes. Chemistry. (2019).
14. Melissa A. Scranton, Joseph T. Ostrand, D. Ryan Georgianna, Shane M. Lofgren, Daphne Li, Rosalie C. Ellis, David N. Carruthers, Andreas Dräger, David L. Masica, Stephen P. Mayfield. Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii. Algal Research. (2016).
15. Merchant, S. S. et al. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Science 318, 245–250 (2007).
16. Oka T, Tooi O, Mitsui N, Miyahara M, Ohnishi Y, Takase M, Kashiwagi A, Shinkai T, Santo N, Iguchi T. Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis. Aquat Toxicol. (2008).
17. Ralston-Hooper K, Hardy J, Hahn L, Ochoa-Acuña H, Lee LS, Mollenhauer R, Sepúlveda MS. Acute and chronic toxicity of atrazine and its metabolites deethylatrazine and deisopropylatrazine on aquatic organisms. Ecotoxicology. (2009).
18. Rusch M, Spielmeyer A, Zorn H, Hamscher G. Degradation and transformation of fluoroquinolones by microorganisms with special emphasis on ciprofloxacin. Appl Microbiol Biotechnol. (2019).
19. Sadowsky MJ, Tong Z, de Souza M, Wackett LP. AtzC is a new member of the amidohydrolase protein superfamily and is homologous to other atrazine-metabolizing enzymes. J Bacteriol. (1998).
20. Sánchez OF, Lin L, Bryan CJ, Xie J, Freeman JL, Yuan C. Profiling epigenetic changes in human cell line induced by atrazine exposure. Environ Pollut. (2020).
21. Schroda M. Good News for Nuclear Transgene Expression in Chlamydomonas. Cells. (2019).
22. de Souza ML, Sadowsky MJ, Wackett LP. Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. J Bacteriol. (1996).
23. de Souza ML, Wackett LP, Sadowsky MJ. The atzABC genes encoding atrazine catabolism are located on a self-transmissible plasmid in Pseudomonas sp. strain ADP. Appl Environ Microbiol. (1998).
24. Tilbrook K, Dubois M, Crocco CD, Yin R, Chappuis R, Allorent G, Schmid-Siegert E, Goldschmidt-Clermont M, Ulm R. UV-B Perception and Acclimation in Chlamydomonas reinhardtii. Plant Cell. (2016).
25. Ulm R, Jenkins GI. Q&A: How do plants sense and respond to UV-B radiation? BMC Biol. (2015).
26. Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave KA, Rossner MJ. Monitoring regulated protein-protein interactions using split TEV. Nat Methods. (2006).
27. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. (2011).
28. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S. Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng Bugs. (2012).
2. Baier T, Wichmann J, Kruse O, Lauersen KJ. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Res. (2018).
3. Christie JM, Arvai AS, Baxter KJ, Heilmann M, Pratt AJ, O'Hara A, Kelly SM, Hothorn M, Smith BO, Hitomi K, Jenkins GI, Getzoff ED. Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science. (2012).
4. Cloix C, Kaiserli E, Heilmann M, Baxter KJ, Brown BA, O'Hara A, Smith BO, Christie JM, Jenkins GI. C-terminal region of the UV-B photoreceptor UVR8 initiates signaling through interaction with the COP1 protein. Proc Natl Acad Sci U S A. (2012).
5. Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, Pérez-Pérez ME, Auroy P, Gorchs Rovira A, Sauret-Gueto S, Niemeyer J, Spaniol B, Theis J, Trösch R, Westrich LD, Vavitsas K, Baier T, Hübner W, de Carpentier F, Cassarini M, Danon A, Henri J, Marchand CH, de Mia M, Sarkissian K, Baulcombe DC, Peltier G, Crespo JL, Kruse O, Jensen PE, Schroda M, Smith AG, Lemaire SD. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth Biol. (2018).
6. Daniel Jaeger, Thomas Baier, Kyle J. Lauersen. Intronserter, an advanced online tool for design of intron containing transgenes. Algal Research. (2019).
7. Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. (2008).
8. Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S. A golden gate modular cloning toolbox for plants. ACS Synth Biol. (2014).
9. Govantes F, García-González V, Porrúa O, Platero AI, Jiménez-Fernández A, Santero E. Regulation of the atrazine-degradative genes in Pseudomonas sp. strain ADP. FEMS Microbiol Lett. (2010).
10. Heins JN, Suriano JR, Taniuchi H, Anfinsen CB. Characterization of a nuclease produced by Staphylococcus aureus. J Biol Chem. (1967).
11. Hom-Diaz A, Llorca M, Rodríguez-Mozaz S, Vicent T, Barceló D, Blánquez P. Microalgae cultivation on wastewater digestate: β-estradiol and 17α-ethynylestradiol degradation and transformation products identification. J Environ Manage. (2015).
12. Jablonowski ND, Schäffer A, Burauel P. Still present after all these years: persistence plus potential toxicity raise questions about the use of atrazine. Environ Sci Pollut Res Int. (2011).
13. Klewer L, Wu YW. Light-Induced Dimerization Approaches to Control Cellular Processes. Chemistry. (2019).
14. Melissa A. Scranton, Joseph T. Ostrand, D. Ryan Georgianna, Shane M. Lofgren, Daphne Li, Rosalie C. Ellis, David N. Carruthers, Andreas Dräger, David L. Masica, Stephen P. Mayfield. Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii. Algal Research. (2016).
15. Merchant, S. S. et al. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions. Science 318, 245–250 (2007).
16. Oka T, Tooi O, Mitsui N, Miyahara M, Ohnishi Y, Takase M, Kashiwagi A, Shinkai T, Santo N, Iguchi T. Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis. Aquat Toxicol. (2008).
17. Ralston-Hooper K, Hardy J, Hahn L, Ochoa-Acuña H, Lee LS, Mollenhauer R, Sepúlveda MS. Acute and chronic toxicity of atrazine and its metabolites deethylatrazine and deisopropylatrazine on aquatic organisms. Ecotoxicology. (2009).
18. Rusch M, Spielmeyer A, Zorn H, Hamscher G. Degradation and transformation of fluoroquinolones by microorganisms with special emphasis on ciprofloxacin. Appl Microbiol Biotechnol. (2019).
19. Sadowsky MJ, Tong Z, de Souza M, Wackett LP. AtzC is a new member of the amidohydrolase protein superfamily and is homologous to other atrazine-metabolizing enzymes. J Bacteriol. (1998).
20. Sánchez OF, Lin L, Bryan CJ, Xie J, Freeman JL, Yuan C. Profiling epigenetic changes in human cell line induced by atrazine exposure. Environ Pollut. (2020).
21. Schroda M. Good News for Nuclear Transgene Expression in Chlamydomonas. Cells. (2019).
22. de Souza ML, Sadowsky MJ, Wackett LP. Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. J Bacteriol. (1996).
23. de Souza ML, Wackett LP, Sadowsky MJ. The atzABC genes encoding atrazine catabolism are located on a self-transmissible plasmid in Pseudomonas sp. strain ADP. Appl Environ Microbiol. (1998).
24. Tilbrook K, Dubois M, Crocco CD, Yin R, Chappuis R, Allorent G, Schmid-Siegert E, Goldschmidt-Clermont M, Ulm R. UV-B Perception and Acclimation in Chlamydomonas reinhardtii. Plant Cell. (2016).
25. Ulm R, Jenkins GI. Q&A: How do plants sense and respond to UV-B radiation? BMC Biol. (2015).
26. Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave KA, Rossner MJ. Monitoring regulated protein-protein interactions using split TEV. Nat Methods. (2006).
27. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. (2011).
28. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S. Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng Bugs. (2012).
Acknowledgements & sponsors
We would like to sincerely thank our sponsors for their support, without them our project would not have been possible.
Thanks to their contribution, we were able to carry out lab experiments, promote our project, raise awareness about water environmental issues and improve our work day by day.
A special thanks to Stephane Lemaire’s Laboratory of Computational and Quantitative Biology, that welcomed us for our experiments and the "organic contaminants" (CO) pole of UMR METIS for helping us conduct the analysis of Seine’s water.
Of course, we would like to warmly thank our three PIs: Pierre Crozet, Marco Da Costa and Frederique Peronnet, who helped us throughout this year.
Finally, we would like to thank all the people who helped our project by participating in our crowdfunding campaign on HelloAsso. We are grateful for all the love and support we received.
Thanks to their contribution, we were able to carry out lab experiments, promote our project, raise awareness about water environmental issues and improve our work day by day.
A special thanks to Stephane Lemaire’s Laboratory of Computational and Quantitative Biology, that welcomed us for our experiments and the "organic contaminants" (CO) pole of UMR METIS for helping us conduct the analysis of Seine’s water.
Of course, we would like to warmly thank our three PIs: Pierre Crozet, Marco Da Costa and Frederique Peronnet, who helped us throughout this year.
Finally, we would like to thank all the people who helped our project by participating in our crowdfunding campaign on HelloAsso. We are grateful for all the love and support we received.