iGEM

iGEM UPNavarra 2019

Nowadays, surface and groundwater quality deterioration is considered as one of the most disturbing environmental problems in the world, with major impacts on environment and human health. 

One of the most dangerous water contaminants are heavy metals. Some of them are essential elements for living organisms, but they could become an ecological hazard if exceeding certain thresholds. In this line, there is a growing concern about the human activities that have drastically altered the geochemical cycles of heavy metals, as well as their biochemical balance. The presence of heavy metals in natural environments has seen a drastic increase in recent years [Singh et al., 2011]. Factors empowering this fact in the last century include the technological and industrial development, the consumerism and the enormous production of waste. 

An early effect of the presence of heavy metals in the environment is the degradation of the soil, decreasing its productivity. The accumulation of heavy metals in plants and crops, over long periods of time, can also affect the natural fauna. Last, but not least, prolonged exposure to heavy metals that are toxic or carcinogenic (such as cadmium, lead, nickel, arsenic or mercury), can cause deleterious health effects in humans. For that reason, it is important to avoid the ingestion of food in contact with contaminated waters, since chemical substances used in the agricultural industry sometimes contain heavy metals, contaminating vegetables, fruits and meat for human consumption [Kahn et al., 2008]. 

A paradigmatic case of heavy metal is that of mercury, heavily linked to food consumption. According to the European Environment Agency (EEA), mercury is the most hazardous metal in European rivers, lakes and oceans, mostly due to their ingestion by fishes, which are further consumed by animals and humans. This drafts a clear pathway for mercury ingestion on a daily basis, considering the significant presence of this heavy metal in Europe. 

Nitrate is another problematic substance present in water resources that can cause a negative impact on environment and human health. Actually, diffuse pollution from agriculture is one of the main environmental problems in Europe, responsible for poor water quality. Nitrate levels in water resources have increased specifically in many areas of the world due to the rise of intensive farming, with excessive applications of inorganic fertilizer and animal manure in agricultural areas [Mateo-Sagasta et al., 2017]. 

The accumulation of nitrate in surface and groundwater may cause serious illness to both wildlife and humans. It favours water eutrophication, promoting structural changes to the aquatic ecosystem, with increased production of algae and aquatic plants and depletion of fish species when the oxygen is consumed. At the same time, nitrate intake from drinking water can be direct cause of several health conditions, including methemoglobinemia, specific cancers or birth defects [Ward et al., 2018].

Our region, Navarra, is not alien to this problem. Following the EU Directive 91/676/CEE, addressed to the protection of waters from cross contamination from agricultural activities, different regions were detected and put under supervision for their potential health-damaging situations. Such regions are the hydrological basins of the rivers Cidacos, Robo and Ebro-Aragon. Our local government did not only restrict to comply with EU regulations, also passing regional laws (Orden Foral, in their local naming) 247/2018. This law regulates the control of the areas, including registered supervision and, additionally, established an action plan to be executed in the period 2018-2022. Interestingly, the plan includes a specific section addressed to the scientific dissemination of the problem of water contamination, including public information and spread of good agricultural practices. This dissemination is being taken coordinately with the EU-funded LIFE Concert’eau project.

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Detection and elimination of heavy metals and nitrate from our environment should be, therefore, a priority in our local socio economical context. Our team is concerned about this issue. Hence, our goal for the project is to develop genetically engineered bacterial biosensors to detect and quantify the concentration of heavy metals (cadmium, copper and mercury) and nitrate in water. The biosensors should be able to take different colour intensities depending on the concentration of the target substances. Hence, a mathematical study will be needed to correlate such colour intensities to contamination levels. In practical terms, this should empower the distribution of cheap alert kits composed of (a) simplistic bacterial recipients on which potential contaminated water is to be poured and (b) a cell phone App able to estimate the concentration of contaminants from pictures of the recipient.  Having this kit would allow environmental agents and technicians to autonomously obtain early on-site estimates of water pollution within minutes.

Our team is composed of 15 students who study different scientific degrees pioneers at the university, which are Biotechnology, Sciences, Biomedical Engineering and Data Science, all of them enrolled at the Universidad Publica de Navarra (UPNA). This is the first time the UPNA enlists a team for the iGEM competition. If you want to set out on this journey with us, you can follow us on our social media (Instagram, Twitter and Facebook). We also have a blog where we write about our daily work and some funny anecdotes. If you want to contact us directly, send us a message to our email or any other available social media.



References:

- Singh, R., Gautam, N., Mishra, A., and Gupta, R. 2011. Heavy metals and living systems: An overview. Indian Journal of Pharmacology 43(3), 246–253.

- Kahn, S., Cao, Q., Zheng, Y.M., Huang, Y.Z. and Zhu, Y.G. 2008. Health risks of heavy metals in contaminated soils and food crops irrigated with wastewater in Beijing, China. Environmental Pollution 152(3), 686-692.

- Mateo-Sagasta, J.; Zadeh, S. M.; Turral, H. and Burke, J. 2017. Water pollution from agriculture: a global review. Executive summary. Rome, Italy: FAO; Colombo, Sri Lanka: International Water Management Institute (IWMI). CGIAR Research Program on Water, Land and Ecosystems (WLE). 35p.

- Ward M.H, Jones R.R, Brender J.D, de Kok T.H, Weyer P.J, Nolan B.T, Villanueva C.M, and van Breda S.G. 2018. Drinking water nitrate and human health: An updated review. International Journal of Environmental Research and Public Health 15(7): 1557.




Edited by: Stefano Donati

iGEM Athens 2019: Machine Enhanced Directed Evolution of Aptamers

Imagine a group of molecules so diverse, that they could have applications throughout biology, chemistry, medicine, the environment, and even space studies. Additionally, these molecules could be easily synthesized. Is this a future scenario? Surprisingly, no, and these molecules are called aptamers - small oligonucleotides or oligopeptides that bind specifically to a target molecule, just like antibodies!

Why are aptamers not the bread-and-butter of every scientist then, you may ask? We did begin with that question in mind when we were considering alternatives for our iGEM project, as the freshly-composed iGEM Athens 2019 team. Hundreds upon hundreds of papers about possible applications of aptamers turned up after a simple literature search, but no reason why they are not more widely utilised in science. After a while it became apparent: they are not easily created in the first place.

GEM Athens 2019 group photo: Left to right – Nikolas Karvelas, Iasonas Papadopoulos, Nikolas Kalavros, Theofano Zioumpou. Front row: Alexandra Karvela, Anna Maria Tsiatsiani, Marilena De Pian, Iasonas Milionis, Anastasios Galanis

GEM Athens 2019 group photo: Left to right – Nikolas Karvelas, Iasonas Papadopoulos, Nikolas Kalavros, Theofano Zioumpou. Front row: Alexandra Karvela, Anna Maria Tsiatsiani, Marilena De Pian, Iasonas Milionis, Anastasios Galanis

Aptamers binding to a specific target molecule are synthesized via a method called SELEX (Systematic Evolution of Ligands by EXponential enrichment). While this method has proved to be effective, it requires special equipment, trillions of initial random oligonucleotide sequences for the first selection rounds, and a considerable amount of time until it results in the selection of the aptamer with the best affinity to the target. This can take from a few days, usually, to several weeks. Another important disadvantage of the SELEX method is that, although aptamers can be functional in vitro, they often aren’t in vivο.

Our team decided to propose a novel mechanism of aptamer development, MEDEA - Machine Enhanced Directed Evolution of Aptamers. Inspired by the recent work on directed evolution of proteins and previous iGEM teams such as Heidelberg ‘15 and ‘17, our project aims to create an intracellular platform for the evolution of optimisedaptamers, in E. coli cells. The circuit, along with the sequence of the aptamer, will be carried by a plasmid, enablin g the adaptation of our system for other organisms as well, such as S. cerevisae. But this will have to wait, since our proposed system is the first new method of aptamer development in the last 30 years and, as far as we know, the first intracellular method of functional RNA directed evolution in the literature.

How will the evolution of our aptamer sequence be achieved? Through the interaction of three modules: the module containing the aptamer, the mutagenesis module and the selection module. The aptamer module contains theaptamer sequence, of course, connected to a ribozyme. When the aptamer binds to its target, the ribozyme is activated, cleaving a small sequence off its 3’ end, which also happens to be a STAR - Small Transcription Activating RNA. The STAR will activate the transcription of an antibiotic resistance molecule cassette, consisting of two antibiotic  resistance genes and serving as the selection module. The mutagenesis will be performed by the EvolvR system, a recent discovery in the field of in vivo directed evolution. The EvolvR system is comprised by a Cas9 fused to an error-prone polymerase, thus enabling mutagenesis in a specific sequence. We also plan to test a novel variant with Cpf1 instead of Cas9, in order to optimize EvolvR’s efficiency.

The logic behind our design is simple: when the aptazyme binds to the target molecule, antibiotic resistance is achieved. As aptamers evolve, bacteria that carry the best ones will exhibit better ability to adapt to elevated antibiotic concentrations, and the other will die. A few cycles will result in an optimized aptamer sequence.

The MEDEA pipeline for aptamer production, consisting of both a Dry and a Wet Lab part, and the advantages it will offer to researchers.

The MEDEA pipeline for aptamer production, consisting of both a Dry and a Wet Lab part, and the advantages it will offer to researchers.

Dry Lab also plays a considerable role in our project. If the initial aptamer sequence was totally random, the chances of it binding to unwanted ligands would be high. Our software will ensure that we create highly specific aptamers for our targets. Furthermore, a side-project focuses on the engineering of a cheap and easy-to-use morbidostat for continuous liquid cultures.

A pattern starts to emerge here about the theme of our human practices work, which revolves around the basic concept of Access - to science, to technology, to information. As we first composed our team, we became instantly aware how people in Greece do not have access to the most recent advances in science, and also how difficult it was for someone to get involved in the scientific community in the first place. Therefore we aim to work on ways to promote access to synthetic biology in Greece and beyond!

If you want to know more about our project or aptamers in general, don’t hesitate to contact us via email. You can also follow our Facebook, Instagram and Twitter pages, or we will be glad to meet you directly at the Giant Jamboree in Boston!

First Meet Up of the Greek iGEM teams

iGEM Thessaly: The research team from the University of Thessaly organized the First Meet Up of all the Greek iGEM teams at the city of Larissa on Saturday 13 July. 



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The students of the iGEM Thessaly team conducted from 12 to 14 of July a meeting with the Greek teams iGEM Athens and Thessaloniki that are also taking part in the iGEM competition this year at the city of Larissa, Thessaly. 

iGEM is an international completion taking place in Boston on October. It started in 2004 at MIT with only a few teams participating, exclusively from the United States. Today, there are more than 300 teams competing from all over the world. 

One of iGEM’s main goals is to promote collaboration among the iGEM teams and, as an extension, to society. In this context, iGEM Thessaly hosted the First Greek Meet Up and invited iGEM Athens and Thessaloniki to Larissa. The aim of this meet-up was for every team to present their project to the rest and receive useful feedback, comments, and ideas. The conference that was held on Saturday 13th of July.Apart from the teams’ presentations, an iGEM Alumni panel (consisting of exceptional guests with great experience in the iGEM competition) initiated  a constructive discussion with and filled a lot of gaps for the students that are making their first attempt to participate in something this big. 

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The conference was enriched by interesting talks by postdoctoral researchers of the Department of Biochemistry and Biotechnology. The invited speakers  were Constantine Garagounis, from the Laboratory of Plant and Environmental Biotechnology, and Konstantina Tsoumani , from the Laboratory of Molecular Biology and Genomics., We were also happy to host the coordinator of Academia and Research Committee of After iGEM Thea Chrysostomou, the iGEM Sheffield supervisor Dimitris Michailidis, and Giannis Ntekas, iGEM Athens 2018 team leader. Finally,  it was a great honor that our PI Papadopoulou Kalliope, who has been supporting as from the beginning, attended our meet up. 

We wish good luck to all the Greek iGEM teams! 


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iGEM Thessaly’s research project is being supported by the research infrastructure Omic- Engine, States Scholarship Foundation (ΙΚΥ), Research Committee of the University of Thessaly, Hellenic Petroleum, and ELPEN. 


Contact

iGEM Thessaly

e-mail: igem.thessaly@gmail.com 

website: http://igem-thessaly.uth.gr 

Facebook: https://www.facebook.com/igemthessaly 

Ιnstagram: https://www.instagram.com/igemthessaly 

Twitter: https://twitter.com/igemthessaly 

YouTube: https://www.youtube.com/channel/UCBHXzFL7r9xxHxqQICznphA 

The First Synthetic Biology Conference in Cyprus

AfteriGEM, the University of Nicosia, The Cyprus Institute of Neurology and Genetics, the Cyprus School of Molecular Medicine and the European University of Cyprus joined together to make the first SynBio Conference in Cyprus reality on the 29th and 30th of March, 2019. The conference scope was dedicated to the advancement of synthetic biology, education, and the development of an open community

by Thea Chrysostomou

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Synthetic biology as an emerging interdisciplinary field that focuses on the design and creation of new biological components, as well as the reprogramming of already existing biological systems to function as optimized entities, served as a catalyst and started the national and academic conversation on the possible benefits it can bring to the island. This conference helped us understand the benefits and implications of this encounter between technology and biology, and how SynBio can add value to all aspects of Cyprus and global society in medicine, technology, research, education, environment, economy, agriculture and even art.

Results are so far promising; new biological parts and systems such as tumor-seeking microbes for cancer treatment and photosynthetic systems for fuel production are only the beginning of a series of in-progress developments which have the potential to positively reshape everyday life. 

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Through panel discussions and talks of 25 speakers from around the world, this event gave the Cypriot scientific and business ecosystem the opportunity to push the boundaries of synthetic biology and through the starting line of this movement - the intersection between biology and technology - in the island as well.

After the introduction for the conference in the local TV by the former minister of health, Dr. Stavros Malas and Thea Chrysostomou (EU iGEM Ambassador), the first day of the conference started with Thea and Representatives of Ministry of Health and Ministry of Education addressing the audience, followed by panel discussions.

Dr. Tuck Seng Wong from the University of Sheffield in UK, Dr. Lital Alfonta from the Ben Gurion University in Israel, Dr. Vassily Hadjimanikatis from Ecole Polytechnique Federale de Lausanne (EPFL), Switzerland, Dr. Konstantinos Vavitsas from the University of Queensland, Australia, and Dr. Kostas Mathiopoulos from the University of Thessaly gave us some insights on SynBio communities and iGEM teams regionally, their experiences, educational hubs and what could be some differentiating factors to bring constituents such as academia, research, industry, government involvement for the ecosystem of Cyprus to thrive on this field and be part of this movement.

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Meagan Lizarazo, the Vice President of the iGEM foundation gave a very interesting talk on the history of iGEM and SynBio, vision for the future, startups, success stories in a global level (eg. PvP Biologics, Bluepha).

Prof. Lital Alfonta talked more about her research on “Genetic Code expansion for improved electron transfer”, Prof. Philippos Patsalis about his company NIPD Genetics on “Non-invasive Genetic Tests for Reproductive Medicine and Oncology”, Dr. Yiannis Sarigiannis on “Synthetic Biology as a useful tool in antimicrobial drug discovery”, Dr. George M. Spyrou on “ Systems Bioinformatics and Network Rewiring towards Precision Medicine”,  Dr Margarita Zachariou on “Computational Modelling of Brain/Neural Plasticity”, Dr. Vasiliki Gkretsi on “Targeting metastasis: could Ras Suppressor-1 be the key?”Dr. Kyriaki Michailidou “Large Scale genomics association studies in breast cancer”, Dr. Tuck Seng Wong on “Biological carbon dioxide capture and utilization(bioCCU)”, DR. Vasilly Hatzimanikatis on “What do we need from nature’s chemical toolbox for Synthetic Metabolism?”, Dr. Kostas Mathiopoulos on “Enginnering insects for pest control” and Dr Konstantinos Vavitsas on “ Driving synthetic Biology on with sunlight”.

We also had some after iGEMers and current iGEM teams presenting their experience in iGEM, how it shaped their career, what effect it has on their lives being part of this ecostystem and their research projects. Yiannis Ntekas from the National Technical University of Athens from iGEM 2018 talked about “Toehold switch enabled viral detection via routine glucose monitoring technology”, Fran Quero from the Complutense Univeristy of Madrid, iGEM 2018 and 2019 talked about “ From DIYBio to iGEM. The Spanish example.” Alexis Casas and Antoine Levrier from Bettencourt iGEM team 2018 in Paris on “Cell- free expression platforms enable ne possibilitis at iGEM and beyond”. Chris Graham from the University of Nottingham, iGEM 2017 on “Synthetic Biology in UK, a biological key and how iGEM changes a young scientist’s perspective”. Athina Milona and Thodori Kontogiannis represented iGEM Thessaly 2019 on “ Spot the iGEM impact; a Greek aspect of a worldwide phenomenon”. Dimitrios Michailidis from the University of Sheffield talked about Life after University. My colleague Will Wright talked about afteriGEM and entrepreneurial opportunities in iGEM and Thomas Landrain on his startups: La Paillasse, PILI, and cJOGL.

It is my conviction that the projects presented during this conference have made all of us wealthier in knowledge, ideas, and inspiration.

It is my conviction that the projects presented during this conference have made all of us wealthier in knowledge, ideas, and inspiration.

iGEM Cyprus will participate in 2020 as a research team representing the island in Boston with all the major Unis in the country involved.

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Thea Chrysostomou is currently working for the iGEM Foundation as the European Ambassador. At the same time she is continuing her research on Machine Learning and Biophysics in Paris. She has graduated from the University of Sheffield as a Biomedical Scientist.

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.


By Nympha Elisa M. Sia and Gayetri Ramachandran - iGEM Paris Bettencourt 2018