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.

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.

iGEM Leiden 2018: Fifty Shades of Stress

 A colourful screening platform for detecting bacterial cell stress.  Left to right: Lotte Weel (Human Practices manager), Marijke Grundeken (PR manager), Chiel van Amstel (Programming manager), Laurens ter Haar (Wiki manager), Daphne van den Homberg (Secretary & design manager), Marjolein Crooijmans (Science manager), Charlotte de Ceunink van Capelle (Team captain), Tim de Jong (Fundraising manager), Mees Fox (Event manager). Bottom row: Carli Koster (PR manager), Maaike de Jong (Treasurer), Jazzy de Waard (Fundraising manager), Germaine Aalderink (Lab & Safety manager)

A colourful screening platform for detecting bacterial cell stress.

Left to right: Lotte Weel (Human Practices manager), Marijke Grundeken (PR manager), Chiel van Amstel (Programming manager), Laurens ter Haar (Wiki manager), Daphne van den Homberg (Secretary & design manager), Marjolein Crooijmans (Science manager), Charlotte de Ceunink van Capelle (Team captain), Tim de Jong (Fundraising manager), Mees Fox (Event manager). Bottom row: Carli Koster (PR manager), Maaike de Jong (Treasurer), Jazzy de Waard (Fundraising manager), Germaine Aalderink (Lab & Safety manager)

If you have ever had a bacterial infection, you probably went to the doctor, got an antibiotic treatment for a couple of weeks and that was the end of the story. However, this has not always been the case. Before 1928, when Alexander Fleming discovered antibiotics, thousands of people died annually of bacterial infections. This era could return with the rise of antibiotic resistance and your next bacterial infection may end differently.

While we have been able to end almost all bacterial infections easily for the past decades using antibiotics, more and more pathogenic bacteria are becoming resistant to current therapies. This resistance develops naturally as a result of evolution. Some bacteria may have a genetic advantage which allows them to survive antibacterial treatments. Through natural selection this population will grow, making the bacteria strain resistant. The occurrence of resistance is further increased by inappropriate use and prescription of antibiotics.

Antibiotic resistance is predicted to lead to 10 million annual deaths by 2050 which illustrates the need for new solutions. However, only a handful of new antibiotics have been found in the past three decades. Fortunately, the iGEM team from Leiden University will develop a new approach to this problem. We will develop a system which will enable easy and cheap identification of different kinds of bacterial cell stress. Our system can be implemented in screenings for new antibacterial substances which will help in the fight against resistant bacteria.

To realise this new screening method, we will develop an E. coli reporter strain that produces a variety of colourful chromoproteins when exposed to distinct cellular stresses. To illustrate, bacteria exposed to a substance interfering with protein translation will produce a red colour, whereas cell wall synthesis inhibitors elicit a blue color. This way the stress caused by potential antibiotics can be screened for. This expands current screening methods, which only return compounds that are lethal to bacteria.

These newly found stress inducing compounds, are not deadly by themselves, but a lethal cocktail can be produced when combining multiple compounds that cause different types of stress. Stressful compounds may also be used as additives in existing therapies. This novel strategy opens up many new possibilities for treatment of resistant bacteria, delaying the onset of resistance.

This new screening method could lead to the discovery of new antibiotics, and help treat your future bacterial infections. This way the antimicrobial resistance crisis can be averted and we will not have to return to the world before 1928.

Visit our website for more information on our project and be sure to check out our social media to follow our journey to Boston!

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-        By Carli Koster – PR iGEM Leiden

3 Questions for Dr. Francesca Ceroni

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In our newest edition of the '3 Questions for' interview format, we spoke to Dr. Francesca Ceroni, located at the Department of Chemical Engineering of Imperial College London. As a Junior Research Fellow, she is making use of bacterial and mammalian synthetic biology to make a difference in bioproduction and biomedicine. Additionally, Dr. Ceroni is also active in the iGEM competition, both as a judge as well as a supervisor.

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

I had just graduated in Pharmaceutical Biotechnologies when I met a Professor in the Faculty of Engineering at the University of Bologna
that was looking for an advisor for the Bologna iGEM team.
Synthetic Biology sounded so interesting to me that I decided to give it a try; I was also happy to work with other students in an interdisciplinary environment.
That was it: Synthetic Biology just captured my interest and passion from that moment on and I continued in the field for my PhD, post doc and ... the present.

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

I would suggest to try to work with people you like, that show vision... choose very well the environment, the people, the once you want to deal with.
That will make a difference in your career.


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

I think there are many challenges, but the one I consider the most difficult to tackle is the complexity of biological systems that impacts on the reliable engineering we want to achieve. We have gone a long way into the understanding and characterisation of the systems we work with, but there is a lot to do and so much diversity to take into account as well.

iGEM Athens 2018: Tackling a future epidemic

  iGEM Athens 2018 team. From left to right: Elena, Nelly, Maria, Natalia, Stelios, Yannis, Leda, Panos and Vasilis.

iGEM Athens 2018 team. From left to right: Elena, Nelly, Maria, Natalia, Stelios, Yannis, Leda, Panos and Vasilis.

MERS-CoV (Middle East Respiratory Syndrome Coronavirus) is a Coronavirus, endemic to the Middle East. The virus attacks the human respiratory system and is highly pathogenic, causing a series of non-specific symptoms which complicate the diagnosis. The World Health Organization has declared MERS-CoV as one of the most likely to cause a future epidemic and urges for further research. The mortality rate of humans infected by MERS-CoV is approximately 35%, making on-time diagnosis critical for treatment and epidemic prevention.

iGEM Athens 2018 team aims to develop a molecular diagnostics kit for the detection of MERS-CoV. To meet the existing societal needs, we aim for an easy-to-use, rapid test that is reliable, safe, and usable on the field – even by the untrained.

  MERS-CoV detection kit, as envisioned by iGEM Athens

MERS-CoV detection kit, as envisioned by iGEM Athens

Our detection mechanism is based on the Toehold-Switch technology. Toehold switches are hairpin-shaped riboregulators that precede a protein coding sequence in a synthetic mRNA molecule. The conformation of the switch regulates the expression of the protein; in the absence of a trigger complementary RNA sequence, the switch region folds, inhibiting the binding of the ribosome to the RNA and subsequently the expression of the coding sequence. If the target sequence is present, it binds to the switch region causing its unfolding, allowing the protein production.

Incorporating this mechanism as a DNA construct in a cell-free transcription and translation system creates a robust, genetically engineered circuit that can be used as a biosensor. DNA or RNA segments of viral or pathogenic origin can provoke the unfolding of a specific toehold switch-gene complex and lead to the expression of a reporter protein, indicating the existence of the target segments. It is preferable that the reporter protein produce an easy-to-read signal, such as a change in colour.

We attempt to refine the existing technology further by testing alternative peptides as strong reporters, improving the rapidness and the sensitivity of our diagnostic test. The reporter protein has to be of a relatively small size, alleviating the transcriptional and translational load as much as possible. We will design toeholds activated by MERS-CoV sequences and regulate the expression of an engineered enzyme

The toehold switch design is assisted by bioinformatics tools suggesting the best candidate target sequences, taking into consideration the viral genome, the sample origin and the natural microbiota of the sample. On this scope, we are designing Pre.Di.C.T. (Predictive Diagnostic Custom Toehold-Switch), a user-friendly generalized workflow that will facilitate the design of molecular-diagnostics systems for future research on other viruses.

For more information about our project’s progress you can visit our official website or follow us on social media: Facebook, Twitter, Instagram.

You can read the original post on the PLOS Synbio Blog: http://blogs.plos.org/synbio/2018/07/16/igem-2018-two-synbio-teams-from-greece-are-here-to-leave-their-mark/