iGEM

ODYSSEE: A modular platform for field diagnosis of Tuberculosis

By iGEM Thessaly 2019

 

Can you imagine a world where everyone has unlimited access to healthcare? A world where equal opportunities are guaranteed, despite economic, social or political status, through the collaboration among countries?

 

Well, this is just not wishful thinking. These are some of the goals set by the UN (Sustainable Development Goals) for a better world by 2030. iGEM Thessaly decided to work on contributing to the effort made for the achievement of these goals.

 

iGEM Thessaly is the first team from the Thessaly area of central Greece to participate in the iGEM competition.  We are ten students from different Departments of the University of Thessaly. Our project “OdysSEE” aims for the fight against the communicable disease Tuberculosis (TB), a major threat for populations affected by crises such as refugees.

ODYSSEE_logo.png

Refugees and migrants are entitled to the same universal human rights and fundamental freedoms as all people, which must always be respected, protected, and fulfilled. More than 85% of refugees flee from and stay in countries with a high burden of TB (Kimbourgh et al, 2012). 

 

Despite increases in notifications of TB, progress in closing detection and treatment gaps is slow and large gaps remain. The goals of the World Health Organization’s End TB strategy will not be achieved without new tools to fight TB. 

IMG_4511.jpg


For this reason, we are developing “OdysSEE”, a rapid, reliable and safe test for early diagnosis of Tuberculosis that would be applied in refugee camps in Greece, as well as worldwide, wherever is needed. OdysSEE reflects the challenging journey that refugees are going through and our logo contains a migratory bird every piece of which represents a unique part of our project.

 

The test will work on urine samples. Once the Mycobacterium tuberculosis, that causes the disease, dies in a patient’s lung, it releases DNA fragments (cell-free DNA - cfDNA) into the blood as it breaks down. cfDNA’s small size allows for it to cross the kidney barrier and appear in the urine (Fernαndez-Carballo et al., 2018). The biomarker we selected is the IS6110 gene (1355 bp), which is located in the genome of the Mycobacterium Tuberculosis (MTB) and encodes for a putative transposase. IS6110 belongs to the family of insertion sequences (IS) of the IS3 category and is most commonly used for the detection of MTB because it is highly conserved (Thierry et al., 1990, Thabet S. & Souissi N., 2016).

 

The detection workflow contains 4 steps of amplification of the target gene. It begins with isothermal DNA amplification of the MTB DNA fragment, with the incorporation of two universal sequences, at 5’ and 3’ end respectively. An in vitro transcription of the amplicon follows with the combination of these two steps enabling addition and amplification of a universal trigger sequence, which is transcribed to RNA.

Odyssee_Project Design.png

This trigger RNA enables the in vitro translation of a toehold switch, a biosensor that encodes for a b-lactamase. b-lactamase is an enzyme that hydrolizes cephalosporins including nitrocefin, which then turns from yellow to red. The colorimetric readout will enable naked eye detection of the result. 


Tuberculosis detection is just the beginning. We aim to create a universal tool able to identify other communicable diseases as well. The key component to achieve this is the trigger RNA that is designed by the team’s wet lab and added to the reverse primer for the first step amplification. This can be achieved by just changing the primer set, while keeping the overhangs that contain the universal trigger, as well as the following path the same, thus targeting different pathogenic agents.

IMG_20190618_120334.jpg

The ultimate goal is to supplement conventional diagnostics by providing a modular, universal diagnostic platform for various diseases so that all patients have access to innovative tools and services for rapid diagnosis and care.

Join our journey and stay in touch with us and our project by following us on Facebook, Instagram and Twitter and also by visiting our website. Any feedback for our project is welcomed and can be addressed to us via email at igem.thessaly@gmail.com.

You can support our effort through our crowdfunding platform here.

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

  

References

Kimbrough, W., Saliba, V., Dahab, M., Haskew, C., & Checchi, F. (2012). The burden of tuberculosis in crisis-affected populations: A systematic review. The Lancet Infectious Diseases, 12(12), 950–965.

D Thierry, A Brisson-Noël, V Vincent-Lévy-Frébault, S Nguyen, J L Guesdon, B Gicquel, Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis (1990), Journal of Clinical Microbiology

Thabet, S., & Souissi, N. (2016). Transposition mechanism, molecular characterization and evolution of IS6110, the specific evolutionary marker of Mycobacterium tuberculosis complex. Molecular Biology Reports, 44(1), 25–34.

Fernández-Carballo, B. L., Broger, T., Wyss, R., Banaei, N., & Denkinger, C. M. (2018). Toward the development of a circulating free DNA-Based in vitro diagnostic test for infectious diseases: A review of evidence for tuberculosis. Journal of Clinical Microbiology, 57(4), 1–9.

 

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. 



logo meetup xor.png

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. 

IMG_20190713_181235.jpg

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! 


IMG_7787.JPG

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 

iGEM Aachen 2019: Plastractor

by Alina Egger and Yasmin Kuhn

banner.png

Currently everybody talks about environmental pollution by plastic. But not only big plastic waste, like plastic bottles, are a problem for us, but also microplastic, which e.g. was found in drinking water. Microplastics, particles smaller than 5mm, generated by degradation via wave motion and UV radiation, can work their way into the marine food chain and eventually into the human body.

With our project, we want to approach the microplastic problem. On the one hand we want to produce an easy way to detect micro- and nanoplastics in fluids and differ between different polymers. On the other hand, our project should create an easy way to extract them. Magnetic purification seemed to fit, as it doesn’t require any chemicals or elevated equipment.

Currently there are known magnetic bacteria existing, e.g. Magnetpospirillum gryphiswaldense, which thrive in the sediments of freshwater streams or marine sediments in very low oxygen environments. The most fascinating ability of these bacteria is their capability to produce so called magnetosomes, spherical vesicle-like structures of membrane-coated, biomineralized ferrite monocrystals with an approximate diameter of 45 nm. These are aligned by special cytoskeletal proteins inside the cell body to form little compass needles, which allow the bacteria to orient themselves along the earth’s magnetic field.

We want to develop novel fusion proteins embedded into the vesicular membrane of magnetosomes being able to specifically bind certain polymers, for example polypropylene (PP). They are consisting of a transmembrane domain as well as a variable linker domain and a domain for binding the polymer.

Figure 1: Schematic binding of polypropylene (PP) to the magnetosome mebrane (right) via the constructed fusion protein (left).

Figure 1: Schematic binding of polypropylene (PP) to the magnetosome mebrane (right) via the constructed fusion protein (left).

Figure 2: Fluorescent detection of the bound plastic particle with bound fluorescent markers.

Figure 2: Fluorescent detection of the bound plastic particle with bound fluorescent markers.

Novel fusion proteins embedded into the vesicular membrane of magnetosomes can be developed, able to specifically bind certain polymers, for example polypropylene (PP). For detection purposes there is a fluorescent protein marker inside the fusion protein that marks the polymer particle for fluorescent detection.

Our project aims to make the world a little less “plastic”. We don’t want to build up new plastic but to remove the one already present. Join the fight against microplastic and support us by visiting our website. You can ask us anything via e-mail (igem@rwth-aachen.de) and also follow us on Facebook, Instagram and Twitter to stay in touch with us and our journey to the competition in October.

The 2019 Aachen iGEM team

The 2019 Aachen iGEM team

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

iGEM TU Delft 2018: Advanced Detection of Performance Enhancement

Left to right:  Lisbeth Schmidtchen; Timmy Paez; Gemma van der Voort; Lisa Büller; Jard Mattens; Janine Nijenhuis; Alex Armstrong; Nicole Bennis; Kavish Kohabir; Venda Mangkusaputra; Susan Bouwmeester; Monique de Leeuw

Left to right:

Lisbeth Schmidtchen; Timmy Paez; Gemma van der Voort; Lisa Büller; Jard Mattens; Janine Nijenhuis; Alex Armstrong; Nicole Bennis; Kavish Kohabir; Venda Mangkusaputra; Susan Bouwmeester; Monique de Leeuw

Doping has been an issue for fair sports for many years. Lance Armstrong for example won the Tour the France seven times before he was caught for the use of doping. At the end of 2012 all these seven victories were taken. The cycling world was shocked.

Lisa Büller

Lisa Büller

Athletes are constantly searching for new types of performing enhancement. Rapid advances in gene therapy enable athletes nowadays to inject themselves with performance enhancing genes, like Erythropoietin (EPO) and insulin-like growth factor 1 (IGF-1), in an almost undetectable process called gene doping. The big advantage of the use of gene doping is the easy bypassing of current detection methods. The World Anti-Doping Agency (WADA) is taking the possibility of gene doping seriously. Sooner or later, the sporting world has to deal with the phenomenon of gene doping to control athletic performance enhancement. The big problem is that no suitable detection method has been accepted for official use so far.

The TU Delft iGEM team is trying to tackle this problem by designing a suitable detection method for gene doping. In October our concept will be presented at the biggest international competition on synthetic biology, iGEM (international Genetically Engineered Machine competition). In this competition over 340 teams compete. All teams try to solve a world problem with the use of synthetic biology. This year the TU Delft is designing an efficient, secure and versatile method for the detection of gene doping.

Venda Mangkusaputra

Venda Mangkusaputra

Our novel detection method uses a dxCas9-Transposase fusion protein to target and tag the specific gene doping DNA with adapters. Only the genes with the adaptor will be sequenced with Oxford Nanopore Technologies’ next generation sequencing. With this method we create a method for targeted sequencing, which can be an efficient, secure and versatile for the detection of gene doping.

Currently, it is most likely that for the administration of gene doping, an adenovirus or a plasmid is used. Both methods bring the doping gene in the cell nucleus, where the gene will be expressed. The inserted genes however, do differ from the regular EPO genes present. Because of the size of the gene, introns must be taken out. We therefore search for the exon-exon junctions, which we do using a dxCas9 with a library of different gRNA sequences that cover all different gene doping changes. When the dxCas9 finds an exon-exon junction, the Tn5 transposase will cut the doping gene and add the sequencing adapters (Figure 1). This method for targeted sequencing will make sure that only gene doping DNA will be sequenced.

Figure 1      Schematic project overview

Figure 1 Schematic project overview

Do you want to know more about our project? Please visit our website for more information and sign up for our newsletter. We are always happy to answer questions via e-mail. You can follow us on Facebook and Twitter to stay in touch with our last sprint to the competition in October.

Get on your marks, get set and join us in the fight against gene doping.