Team:Thessaly/Poster

Poster: Thessaly



Amalthea: A Modular Platform for Monitoring Gastrointestinal Health
Presented by Team Thessaly 2020

Lemonia Apocha1, Ifigenia Daskalaki1 , Magdalini Koroxenidou1 Anastasios Messis1 , Venetios Michelioudakis1 , Georgios Mouchtaridis1 Foteini Papadaki1 , Vasileios Stergiou1 , Emmanouil Stylianakis1 , Asteria Tsapadikou1 , Marios Lange2 , Nikolaos Ntelkis2 , Constantine Garagounis3 , Katerina Moutou4 , Kalliope K.Papadopoulou4


1iGEM Student Team Member
2iGEM Team Advisor
3iGEM Team Instructor
4iGEM Team PI

Abstract

Amalthea is a complete, personalized, modular platform, which provides diagnosis, evaluation of the gut flora, and treatment of IBDs. A non-invasive encapsulated detection module, consisting of a genetically engineered bacteria-based system and an electronic system, will identify metabolite deficiencies directly correlated to IBDs, that may lead to malnutrition. Exploiting a bio-electronic interface to enable real-time monitoring on the patient’s smartphone. Based on this personalized data, a biosynthetic module will respond with selective production of the missing metabolites, thus eliminating that nutritional deficit and relieving the patient of the symptoms. Our product is designed according to healthcare experts and international standards, to ensure biosafety.

Introduction

Malnutrition & IBDs

Our team aims to tackle a multifaceted problem; Malnutrition, which is the main cause or the side effect, of chronic diseases. One in three people globally suffer from at least one form of malnutrition: wasting, stunting, vitamin, and mineral deficiency, overweight or obesity, and diet-related non-communicable diseases. The inability to absorb nutrients can lead to chronic diseases [3-4], such as Inflammatory Bowel Diseases (IBDs). IBDs are a multifactorial family of interrelated diseases that affect the human gut, which affects 1 in 1000 people worldwide in 2020 [1].

world map
Inspiration

Friends and family around us help us see the real state of this problem.
Their quotes as IBDs patients:

“There is no normal life when you live it in the toilet.”

“Autoimmune diseases affect a larger portion of the population than we think, and the difficulties they cause in the daily lives of the people who suffer from them are not always obvious.”

For our purpose, previous iGEM projects helped us to form our project design.

  • TU_Eindhoven 2015 virtual diagnosis of Crohn’s Disease
  • NEU_CHINA 2018/2019 ingestible biosensor
  • UFlorida 2018 and Duesseldorf use of SCFAs

The words of the patients, along with the information we had gathered from our surroundings and various scientific sources led to the creation of AMALTHEA.
logo of our team

Methodology

We aim to accomplish the communication between our inner world and the one outside of it, through the understanding of our body’s needs.

In order to do so, we followed one pathway...

design Detections and Therapeutic System

design Golden Braid assembly: GoldenBraid-based vector toolkit, Part Collection

design Modeling, Characterization,
Proof of Concept, Reporter Module


design Analyzing Results and Change our Approach

Design

Detection System
Diagnosis

calprotection image
Calprotectin:

  • A cell-free diagnostic test consisting of engineered aptamers and toehold switches, that can detect salivary Calprotectin
  • Determination of the stage of the inflammation

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    Evaluation of gut microbiota

    For the validation of the precision of the biomarkers for predicting and monitoring response to treatment, we wanted to combine the results of the Calprotectin levels with the evaluation of the gut microbiota, by measuring the levels of SCFAs.


    image
    Capsule
    • Hosting a bio-electronic interface

    • Engineered bacteria for detection of the molecular footprint of gut microbiota

    • Electronic module for digitalization and transmission of the data

    Therapeutic System

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    Production of SCFAs

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    Capsule

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    Biological Circuit

    NOT GATE

    • Logical negation on its input, inverting the initial signal
    • In the absence of SCFAs🡪 Reporter System is expressed
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    Tango Module

    • SCFAs activate the FFAR2
    • Beta-arrestine with TEV protease is recruited, cleaving the TCS
    • LacI is released leading to the inhibition of the expression of eCFP
    not gate image

    Prom Module

    • SCFAs induce the inhibition of the expression of eCFP
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    Tet-off Module

    • TetR inhibits the expression of LacI
    • aTC (simulation of SCFAs), leads to a lac-regulated inhibition of eCFP
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    Reporter Module

    • Tyrosinase converts L-Tyrosine to L-Dopa and L-Dopa quinone
    • Electrons are released
    • Production of Electric current
    not gate image

    Kill switch (Collaboration with the iGEM team of Ohio State University)

    • Production of MazF, toxin-gene is triggered
    • Bacterias’ imminent death
    • Inducers:
    • Arabinose, which is contained in the capsule
    • Cold-shock, due to environmental expose

    not gate image

    Build


    Golden Braid assembly: GoldenBraid-based vector toolkit


    GoldenBraid assembly strategy

    Following the GoldenBraid assembly strategy that is used for Plant Synthetic Biology, allowed us to increase the multigene assembly efficiency. This strategy is based on Type IIS restriction enzymes allowing the creation of our genetic circuits or our Modules. GB parts that are used are grouping together, performing a defined function (e.g. a complete promoter or a full coding region) in a single DNA element. In order to accomplish the special orientation and arrangement, there are two defined levels of plasmids, due to the use of specific restriction enzymes, the α-level and Ω-level plasmids [2].


    Golden Braid-based vectors toolkit

    We designed a GoldenBraid-based vector toolkit aiming to adjust the GoldenBraid DNA assembly method for heterologous expression of proteins in bacteria, since it was originally developed for plants. For this purpose, we created specifically customized α (alpha) and Ω (omega) GB vectors, using the SEVA plasmid collection, to allow modular cloning and expression in bacteria.



    Part Collection

    We modified our basic DNA parts designing compatible sites, in order to combine them and create level 0 ‘building parts and further assembly into transcription units and circuits into level α & Ω destination vectors. Our final constructs consist of a promoter, a CDS, and a terminator and our final Modules with this repeated pattern. In that way, we built an increasingly complex multigene System that can facilitate the heterologous expression of proteins in bacteria.

    We created a Part Collection, containing all required vectors for basic part domestication and multigenic construct assembly. To prove the power of modular cloning, we assembled 17 Level 0 and 16 higher-order constructs.

    We have submitted a list of Biobricks, which indules our Level 0, Level 1 and Level 2 GBparts and cloning vectors for our GoldenBraid-based vector toolkit.

    Test
    Modeling

    To predict the behavior of our genetic modules, we created a functional model using mathematical logarithms to detect Short-Chain Fatty Acids (SCFAs). We simulated the conditions of our NOT-GATE using the structure of the Tango Module in a simplified way approaching the structure of all our Modules.

    The production of eCFP is induced in the absence of SCFAs

    To predict the physical modeling of the antenna we calculated the impedance behavior and radiation characteristics for a compact flexible antenna operating at the ISM frequency bandwidth (433.05; 434.79 MHz).

    For the ingestible capsule, we built a 3D model of the signal transduction circuit to test its efficacy. This 3D graph of the radiation pattern shows that our antenna can transmit effectively on the X and z axes, which fit well with our project.


    Experiments

    Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent the standard deviation of biological replicates.


    Proof of Concept

    • To demonstrate our system’s functionality, we chose to work with the Prom Module. We assembled SCFA-inducible NOT-GATE constructs and characterized them. To enable inhibition of the expression of eCFP we added 2mM of SCFAs, which showed optimal fluorescence during our characterization experiments.
    Characterization
    • We chose to characterize pFliC. Τo have an extensive view of its functionality, we first characterized its activity at different time-points using two different SCFAs, acetate and propionate, a five-orders-of-magnitude range of concentrations and three reporter genes.
    Reporter Module
    • To test the functionality of the Reporter Module, we set up a plate-reader assay. Tyrosinase converts tyrosine to L-DOPA and L-DOPA quinone leading to melanin production, which absorbs at 400nm.Bacterial Growth after 6h at 30oC incubation with Tyrosinase Assay Buffer.


    Learn/ future directions

    Model:

    • More detailed and efficient modeling
    • Revision of the parameters
    • Testing different models to simulate our genetic and electronic systems

    Characterization:
    • Different reporter genes
    • Lower SCFAs concentration levels
    • Various time-points
    • More acids

    Proof of Concept:
    • Testing our Module for more acids
    • Wider concentration range

    Reporter Module:
    • Inducible promoter

    Education

    1. We wanted to be able to see the problems that patients face, through their own eyes.

    So, patients from the General Hospital of Larissa, suffering from IBDs, completed the questionnaires that we designed. We adhered to all legal formalities and any personal data will remain confidential.



    2. To better understand IBDs, we created a theatrical play, in which all the children from a

    local theater participated, developed to help everyone better understand how important the role of nutrition is and what is caused in the body of children suffering from IBDs.



    3. IBD includes a group of autoimmune diseases and, thus, it is considered a chronic

    disease. Unfortunately, most people in our country, Greece, tend to hide their condition due to the fear of pitifulness and society’s lack of affection and understanding. So, we designed a poster to boost people's morale. This is also a collaboration work with the iGEM CSMU and NKCU teams from Taiwan.



    Conclusion & Achievements
    Conclusion:

    Amalthea provides full monitoring of the gastrointestinal (GI) tract’s health by accomplishing diagnosis, evaluation of the gut microbiota, and treatment of IBDs. The non-invasive encapsulated detection device aims to identify metabolite deficiencies directly correlated to IBDs, exploiting a bio-electronic interface to enable real-time monitoring on the patient’s smartphone and help them achieve their well-being.

    Achievements:

    • Designed: Novel approach for the detection of the function of gut microbiota that guarantees
      • A non-invasive method of diagnosis and evaluation
      • Real-time monitoring
      • Personalized treatment and the body’s response to it
      • Use on a regular basis causing no discomfort to the patient and
      • with no limits to the evaluation of the GI tract

    • Contributed: A GoldenBraid-based vector toolkit and a Part Collection to the iGEM registry to help future iGEM teams
    • Characterized: pFliC, understood its behavior and suggested a better approach to test its functionality
    • Introduced: A novel Reporter Module that converts the biological information to an electrochemical signal

    Acknowledgements and Sponsors

    Acknowledgments

    Lab and general support

    Αfroditi Katsaouni, Alexandros Belavilas, Antonios Giakountis, Athanasios Dalakouras, Athina Milona, Eleftheria Moustaka, Georgios Skretas, Georgios Stamatopoulos, Leandros Tsiotos, Leonidas Anthopoulos, Maria Feka, Maria Gerontopoulou ,Rafail Angelakopoulos,Tilemachos Dimitriou,Vasiliki Kavvatha.

    References

    1. Crohn's & Colitis Foundation, (2020), TAKE ACTION FOR WORLD IBD DAY!,

    https://www.crohnscolitisfoundation.org/WorldIBDDay


    2. Sarrion-Perdigones, A., Vazquez-Vilar, M., Palací, J., Castelijns, B., Forment, J.,

    Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2013). GoldenBraid 2.0: a

    comprehensive DNA assembly framework for plant synthetic biology.
    Plant Physiology, 162(3), 1618–1631.
    https://doi.org/10.1104/pp.113.217661


    3. World Health Organization, Malnutrition, key facts (1 April 2020).

    https://www.who.int/news-room/fact-sheets/detail/malnutrition


    4. World Health Organization. (‎2016)‎. The double burden of malnutrition: policy brief.

    World Health Organization.

    https://apps.who.int/iris/handle/10665/255413.

    License: CC BY-NC-SA 3.0 IGO