SynBioS - towards stronger international connections in synthetic biology

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Accompanying adolescence of the discipline of synthetic biology, the past five years have seen many local, national, and supranational synthetic biology groups founded around the globe. United in the aims of promoting synthetic biology research as well as professional and policy development, the associations can benefit substantially from forging and maintaining strong horizontal connections.

On October 23rd, representatives from six national and supranational synthetic biology associations - EUSynBioS (Europe), SynBio UK (United Kingdom), GASB (Germany), SynBio  Australasia (Oceania), SynBio Canada (Canada), and EBRC SPA (United States of America) came together at the 2018 EUSynBioS Symposium Toulouse to set the foundation for a new international collaborative effort, the SynBioS Consortium. The representatives introduced their history, activities, and future plans through short presentations and discussed various topics of mutual interest, such as funding, social media, and science policy.

Concluding the workshop, the representatives confirmed their interest in continuing discussions as part of the future SynBioS Consortium, which will include regular online meetings focused on exchanging advice, coordinating initiatives, and reviewing progress.

We are looking forward to advancing synthetic biology together and encourage other national synthetic biology associations to join our endeavour.

  • EUSynBioS, SynBio UK, GASB, SynBio Australasia, SynBio Canada, EBRC SP

From Asilomar to Toulouse – Bringing Researchers Together and Synthetic Biology to the Forefront

In frosty February 1975, molecular biologists gathered at Asilomar (California, USA) for a conference going down in history. Following the recombinant DNA revolution, the ethical usage of recombinant DNA in research was discussed. Many aspects of this gathering foreshadowed issues that the child of recombinant DNA technology, synthetic biology, is struggling with nowadays. Asilomar helped shape recombinant DNA technologies and bring them into the public eye by bringing together the researchers involved in this topic.

In sunny October 2018, about 100 synthetic biologists from all over the world gathered in Toulouse, from Master’s students all the way up to distinguished professors and leaders in the field of synthetic biology. In a meeting jointly organized by the European Association of Synthetic Biology Students and Post-docs (EUSynBioS) and the French Research Group on Synthetic and Systems Biology (BioSynSys) at the Institut National des Sciences Appliquées de Toulouse (INSA Toulouse), scientists had the chance to engage in fascinating presentations and discussions with their peers. This joint meeting was a first for both organisations and has shown the potential of collaboration between local and international scientific organisations to foster connection, exchange of ideas, and collaborations.

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During four days, leaders of synthetic biology such as Prof. Dr. Sven Panke from ETH Zurich, Prof. Dr. Beatrix Suess from TU Darmstadt or Prof. Dr. Jérôme Bonnet from the University of Montpellier explained their latest advances in very diverse areas of synthetic biology to the audience. Additionally, many young researchers had the chance to present their research in oral presentations and posters. Led by the idea of a circular bioeconomy powered by synthetic biology, which was illustrated by a keynote presentation by Dr. Lorie Hamelin and an open discussion with leaders in the field. This meeting in Toulouse gave to young as well as to established researchers a potential way forward in our climate change-endangered world.

Another way forward was illustrated in workshops conducted during the conference. Dr. Konstantinos Vavitsas discussed the important longstanding issue of standards in synthetic biology with the participants, Nadine Bongaerts prepared them for conversations with the public through science communication and Dr. Pablo Ivan Nikel led a career development workshop to ensure the success of the young researchers present. Accompanied by delicious French food & wines, our participants thus had plenty of exciting science around them, which would have turned the Asilomar participants green with envy!

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Yet there is an additional parallel with conferences such as Asilomar: organization, representation and the determination to bring the topic into the public eye. Next to EUSynBioS, national associations for synthetic biology such as the German Association for Synthetic Biology (GASB), Synthetic Biology Canada (SynBio Canada), Synthetic Biology Australasia (SBA), Synthetic Biology UK (SynBio UK) and the US-based Engineering Biology Research Consortium Student and Postdoc Association (EBRC SPA) also presented their organizations and plans for the future. With the aim of constructing a worldwide SynBioS Consortium to help coordinate initiatives and strengthen the ties between countries and continents, the national associations exchanged information and engaged in fruitful discussion. Analogous to Asilomar, meetings such as this symposium in Toulouse helps to shape the development of synthetic biology, both within as well as without by modulating its interaction with the general public surrounding it. This is particularly important nowadays, in a world endangered by climate-change and in which scientists and synthetic biologists need to bring forward new solutions to solve humanities’ most pressing challenges.

After four days of intense engagement on every level, the participants travelled back to their respective countries, enriched in knowledge, connections, and experiences. If our participants have even a fraction of the satisfaction we have with the event then we can consider it as a major success. See you at the next synthetic biology symposium!

Posted by courtesy of the PLOS Synbio Community blog, where this was originally published.

   Daniel Bojar    and    Adam Amara    are EUSynBioS steering committee members.    Alicia Calvo-Villamañán    is a member of the student committee at BioSynSys.

Daniel Bojar and Adam Amara are EUSynBioS steering committee members. Alicia Calvo-Villamañán is a member of the student committee at BioSynSys.

iGEM Paris Saclay 2018: MethotrExit - a HeteroGenious Cleaning Factory

Cytotoxic anticancer drugs are harmful chemicals found in hospital wastewater at high concentrations. Physical and chemical degradation methods exist but are often inefficient, unsustainable or expensive. We propose MethotrExit, a bioreactor-based approach to tackle this problem. We focused on the biotransformation of methotrexate (MTX), a widely used anticancer drug.

After choosing an appropriate chassis strains, we designed synthetic cassettes encoding a new biotransformation pathway using a heterologous carboxypeptidase in Escherichia coli. In only 5 hours, MethotrExit drastically removes MTX from the media. However, the degradation of anticancer drugs and the biotransformation pathway itself can be toxic. To overcome these issues, Biobricks bringing heterogeneity in enzyme expression were built to ensure the survival of a subpopulation. Modeling of this system highlights the interest of a division of labor between ‘cleaning’ and ‘stem’ bacterial cells.

 From left to right, in the back: Yueying Zhu, Ousman Bao, Guillaume Garnier, Kenn Papadopoulo, Arthur Radoux, Julie Miesch, William Briand, Britany Marta, Clémence Maupu. In the front: Raphaël Guegan, Julie Rojahn, Mahnaz Sabeti Azad (advisor), Céline Aubry (advisor), Stéphanie Bury-Moné (instructor). Advisors not present on the picture: Phillipe Bouloc, Sylvie Lautru, Olivier Namy, Arnaud Ferre

From left to right, in the back: Yueying Zhu, Ousman Bao, Guillaume Garnier, Kenn Papadopoulo, Arthur Radoux, Julie Miesch, William Briand, Britany Marta, Clémence Maupu. In the front: Raphaël Guegan, Julie Rojahn, Mahnaz Sabeti Azad (advisor), Céline Aubry (advisor), Stéphanie Bury-Moné (instructor). Advisors not present on the picture: Phillipe Bouloc, Sylvie Lautru, Olivier Namy, Arnaud Ferre

Choice of an appropriate chassis for the ‘Cleaning Factory’

Escherichia coli is a good chassis since it naturally expresses the AbgT permease which imports folate analogs such as MTX (Green J. 2002). After analysing the MTX-sensitivity of several E. coli K12 WT and efflux pump mutants, we decided to choose both the WT and ΔtolC strains as chassis. Indeed the former presents a strong MTX-resistance (up to 1264 µM MTX) while the latter is MTX-sensitive but devoid of MTX efflux pump. This may limit MTX efflux and favor MTX biotransformation within the cell.

Design of the MTX biotransformation pathway

We focused our attention on two enzymes, Pseudomonas carboxypeptidase G2 (CPG2 or ‘glucarpidase’) and a folylpoly-γ-glutamate synthetase (FolC) (Figure 1.A). CPG2 is the key enzyme that rapidly converts MTX into less toxic metabolites glutamate and DAMPA (2,4-diamino-N10-methylpteroic acid-d3), (Widemann, Sung et al. 2000), (Larimer, Slavnic et al. 2014). We also tested the interest of co-expressing FolC that may enhance MTX catabolism by coupling MTX to polyglutamate (Chabner, Allegra et al. 1985) (Kwon, Lu et al. 2008).

The biotransformation of MTX was monitored by HPLC analysis and bioassays using the E. coli K12 acrA1 mutant as an indicative strain. In only 5 h of incubation with our ‘Cleaning Factories’, MTX was nearly completely removed from LB medium (Figure 1.B).

  Figure 1 :  A) The MTX-biotransformation pathway and division of labor strategy.  B) HPLC analysis of MTX medium incubated with a ‘MTX-Cleaning Factory’. LB medium containing MTX (512 µM) was incubated during 5 h at 37°C with control bacteria (E. coli K12 pSB1C3-tet (BBa_R0040)) or with one of our ‘MTX Cleaning Factory’ (E. coli WT pSB1C3-folC-cpg2 (BBa_K2688009)). The supernatants were filtrated and analysed by HPLC with a reverse phase C18 column. Detection was made using UV spectrophotometry at 303 nm.  C) The ‘HeteroGenious’ device. Interplay between Ler and H-NS for the modulation of LEE5 promoter activity; Fluorescent microscopy of E. coli K12 harboring pSB1C3-LEE5_GFP_native (BBa_K2688012) cultured in LB at 37°C during 24 h.

Figure 1:

A) The MTX-biotransformation pathway and division of labor strategy.

B) HPLC analysis of MTX medium incubated with a ‘MTX-Cleaning Factory’. LB medium containing MTX (512 µM) was incubated during 5 h at 37°C with control bacteria (E. coli K12 pSB1C3-tet (BBa_R0040)) or with one of our ‘MTX Cleaning Factory’ (E. coli WT pSB1C3-folC-cpg2 (BBa_K2688009)). The supernatants were filtrated and analysed by HPLC with a reverse phase C18 column. Detection was made using UV spectrophotometry at 303 nm.

C) The ‘HeteroGenious’ device. Interplay between Ler and H-NS for the modulation of LEE5 promoter activity; Fluorescent microscopy of E. coli K12 harboring pSB1C3-LEE5_GFP_native (BBa_K2688012) cultured in LB at 37°C during 24 h.

Ensuring the maintenance of the bacterial population – towards the ‘HeteroGenious Cleaning Factory’

We observed that the chassis strains harboring both cpg2 and folC expression cassettes present a slight growth delay. Indeed, drug degradation pathways may be associated with a fitness cost. Therefore, we wanted our bioreactor to harbor a heterogeneous synthetic transgene expression. Only two parts are required to implement this ‘HeteroGenious’ system in E. coli: Ler (‘LEE encoded regulator’) and its target LEE5 promoter (Figure 1.C) (Leh, Khodr et al. 2017). The competition between Ler and H-NS (naturally present in E. coli) for LEE5 binding can generate a heterogeneous transgene expression. Modeling a heterogeneous expression of the synthetic pathway within the ‘cleaning factory’ population highlights the interest of a division of labor between ‘cleaning’ and ‘stem-like’ bacterial cells.

Conclusion

We obtained E. coli strains that efficiently remove MTX from culture medium, and a two-part device that can generate heterogeneity of transgene expression within a bacterial population. This study opens new insight concerning the design of ‘Cleaning Factories’. Moreover, E. coli strains able to degrade MTX could be of potential interest as probiotics to treat MTX intoxication.




References

Baba T. et al. (2006) Mol Syst Biol 2: 2006 0008

Chabner B. A. et al. (1985) J Clin Invest 76(3): 907-912

Green J. N. B. et al. (2002) Proceedings of the 12th International Symposium on Pteridines and Folates, National Institutes of Health, Bethesda, Maryland, June 17–22, 2001

Kwon Y. K. et al. (2008) Nat Chem Biol 4(10): 602-608

Larimer C. M. et al. (2014) Adv Enzyme Res 2(1): 39-48 

Leh H. et al. (2017) MBio 8(4)

Widemann B. C. et al. (2000) J Pharmacol Exp Ther 294(3): 894-901.



3 Questions for Prof. Barbara Di Ventura

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In our most recent iteration of the ‘3 Questions For’ interview format, we speak with Prof. Barbara Di Ventura at the BIOSS Centre for Biological Signalling Studies at the University of Freiburg, Germany. Her group is pushing the boundaries of optogenetics and uses light-regulated methods to study cell division and chromosome segregation in bacteria. Herein, and in general, the Di Ventura lab has a strong focus on protein dynamics.

When and why did you move into the field of synthetic biology?

I would say 2002 is the year that marks my entry into the field of synthetic biology. After I graduated in Computer Engineering at the University of Rome “La Sapienza” I moved to Heidelberg to start my PhD at the EMBL in the group of Luis Serrano. At the beginning, the idea was for me to do only mathematical modeling of biological processes. After some months, however, Luis told me I should rather learn to do experiments on my own not to have to wait for others to give me data to model. That’s when I came into the field of synthetic biology, as the project I selected dealt with the transplantation of the p53-Mdm2 module into yeast to study its properties as an oscillator. I think that for someone like me who trained as an engineer, synthetic biology is the most natural way of entering into molecular biology. Eventually we are engineering cells instead of cars or buildings! And we do use computers a lot.


What is the single most important piece of advice that you would give to a current PhD student or a post-doc?

To those working in the field of synthetic biology I would say: take the chance to make the world a better place! To those not working in the field of synthetic biology I would say: what are you waiting for? Join synbio!!


In which areas do you see the main challenges and opportunities for synthetic biology?

The first area that comes into my mind is medicine. There is so much that synthetic biology can do here. The most intriguing to me is the transformation of the concept of treatment from “taking a drug” into “having a synthetic circuit monitor your state of health and react in case anything goes wrong”. Of course, the challenges are many since we need to break down the natural – and understandable – barrier of fear and skepticism that surrounds the idea of introducing something man-made (yet living!!) into our bodies. Moreover, we surely need to go down a long way to make sure that this method is safe and effective. Beyond medicine, synthetic biology can bring a revolution in so many other fields – energy, food production, environment, just to name a few.

iGEM Paris Bettencourt 2018: STAR CORES - Protein scaffolds for star-shaped AMPs

 iGEM Paris Bettencourt Team (2018) group photo. Left to right: Maksim Baković, Juliette Delahaye, Annissa Améziane, Santino Nanini, Elisa Sia (Team leader), Antoine Levrier, Jake Wintermute (Secondary P.I.), Ariel Lindner (Primary P.I.), Darshak Bhatt (Team leader), Oleksandra Sorokina (Advisor). Bottom row: Anastasia Croitoru, Camille Lambert, Naina Goel. Missing from the photo are Shubham Sahu, Alexis Casas, Haotian Guo (Mentor), Ana Santos (Mentor), and Gayetri Ramachandran (Mentor)

iGEM Paris Bettencourt Team (2018) group photo. Left to right: Maksim Baković, Juliette Delahaye, Annissa Améziane, Santino Nanini, Elisa Sia (Team leader), Antoine Levrier, Jake Wintermute (Secondary P.I.), Ariel Lindner (Primary P.I.), Darshak Bhatt (Team leader), Oleksandra Sorokina (Advisor). Bottom row: Anastasia Croitoru, Camille Lambert, Naina Goel. Missing from the photo are Shubham Sahu, Alexis Casas, Haotian Guo (Mentor), Ana Santos (Mentor), and Gayetri Ramachandran (Mentor)

Microorganisms such as bacteria and yeasts are fascinating! They are both beneficial and harmful to us. Over the decades, we have been using antibiotics to kill such harmful, disease-causing bacteria. With time, over-prescription and misuse of these drugs have made bacteria resistant to them; thus, evolving into what we call “superbugs”. This is a global health crisis that we currently face where simple and treatable bacterial infections have become incurable.
Recent statistics have shown that antibiotic resistance is responsible for an estimate of 25,000 deaths per year in the European Union (EU). More importantly, it is predicted to be responsible for up to 700,000 deaths each year, which is expected to rise – overtaking cancer by 2050. Not only does it take many lives but it also has a huge economic impact. In 2009, the cost of treating multidrug-resistant bacterial infections amounted to € 1.5 million in the EU alone. Likewise, according to a CDC report in 2013 entitled, “Antibiotic Resistance Threats in the United States”, antibiotic resistance was responsible for $20 billion in direct health-care costs in the United States.

In order to fight this catastrophe, many strategies have been developed but are primarily focused on humans. Thus, the World Health Organization (WHO) has come up with a more holistic approach to deal with this problem - One health concept. This states that the dispersal of resistance genes is not only limited to human species but it also spread through animals and the environment. Given the complex interactions between different sectors, one has to expand our focus to other areas like animal farming, agricultural industries, hospitals, urban and rural sectors to curb the spread of this man-made problem.
    
In response, the iGEM Paris Bettencourt 2018 team has decided to concentrate on animal-husbandry. According to Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (ANSES), the pig and pork industry consumed the highest amount of farm antibiotics in the year 2015 (129 kg/PCU), making the situation worse, while the European Food and Safety Authority (EFSA) stated that it is time to Reduce, Replace, and Re-think the use of antimicrobials given to animals. Thus, our team chose to work for a possible replacement for antibiotics for reared pigs, along with engaging the public to increase awareness of the misuse of antibiotics.

After doing some intensive research, we came across a promising alternative to antibiotics which is called antimicrobial peptides (AMPs), a diverse class of naturally occurring proteins. AMPs have a broad host range and are highly efficient: since they target the bacterial membranes, resistance to AMPs evolves at a much slower rate. Despite their great potential, there are some limitations for clinical usage including their potential toxicity, susceptibility to protease degradation, and high cost of production.

Considering the prospects of using AMPs and to overcome their drawbacks, we have proposed to employ an E. coli-based cell-free expression platform to produce naturally occurring or artificially designed AMPs (Fig. 1, step 2). These AMPs have been fused to self-assembling scaffold proteins to improve their bactericidal efficiency. We started with screening the best possible AMPs and scaffold protein complexes, in terms of their bactericidal property and biocompatibility (Fig. 1, step 1), which will followed by their production in cell-free systems. Then, we would test their mechanism of action by liposome leakage assay and microscopy. In addition, we want to mathematically model the influence of the charge distribution on the efficacy of the AMPs. To achieve this aim, we have generated 12,000 variants from 5 native sequences which were suitable candidates for designing our library. Lastly, the selected AMPs fused with the scaffold proteins would be tested for their efficacy via killing kinetics experiment in which the minimum inhibitory concentration was also determined (Figure 1, Step 3). We also check for any resistance development after exposure to the controls and our experimental product. Finally, we would determine their toxicity on mammalian cell lines.

 Figure 1. The three core components of the project. Step 1: Screening of the AMPs and scaffold protein complexes. Step 2: Production of the selected AMPs and scaffold protein via E. coli-based cell-free expression. Step 3: Test for the efficacy and safety of the AMPs fused with the scaffold protein produced via cell-free synthesis.

Figure 1. The three core components of the project. Step 1: Screening of the AMPs and scaffold protein complexes. Step 2: Production of the selected AMPs and scaffold protein via E. coli-based cell-free expression. Step 3: Test for the efficacy and safety of the AMPs fused with the scaffold protein produced via cell-free synthesis.

Our battle against antibiotic resistance bugs has just started, and if you would like to join us on our journey to save the planet, please do follow us on our social media portals (Facebook, Instagram, Twitter, and our YouTube channel). Feel free to message us via email or any of our social media accounts. We are open to questions, suggestions, collaborations, and monetary support.