Introducing: GutFeeling

Did you know that:

• The average medication adherence in developed countries is only 50%?
• In Europe, low medication adherence is linked to ~200.000 annual deaths?
• Medical costs for patients who are not adhering to their treatment are of ~125 billion euros in Europe alone?

Our solution:

Our goal is to develop a self-sustained and controlled drug delivery system by engineering the commensal gut bacterium Pseudomonas alcaligenes to autonomously produce and secrete therapeutic compounds in the zebrafish gut, providing a novel strategy for consistent and reliable treatment of chronic diseases such as Parkinson’s Disease.

Our approach

1. Engineered function

We will use standardized molecular cloning techniques such as Golden Gate Assembly to construct our plasmids. We aim for a design that is replicated efficiently in E. coli and functionally transcribed and translated in P. alcaligenes.

Our goal is to make a modular plasmid that allows for various functionalities and safety features. This plasmid will include:

• A constitutive or inducible promoter
• A secretion signal peptide fused to a reporter
• A kill switch and autoregulatory element to ensure safety and dosage control
• An antibiotic resistance marker
• A reporter tag or fusion to enhance protein tracking and diffusion in biological systems

         2.  FPS in the gut: proof of concept

Zebrafish will take up any bacteria added to their aquatic environment into their intestinal system. To show that the bacteria actually stay in the gut, we will use fluorescent proteins that mark the bacterial membrane, as well as a fluorescent marker fused to the secreted product.

          3.  Transwell system

To evaluate whether or not the produced L-dopa can cross the intestinal barrier, we will use an in vivo transwell assay. In this system, Caco-2 human colon cells are combined with mucus producing cells to accurately simulate the gut environment. Culturing the P. alcaligenes in this system will allow analysis of the bacterial L-dopa production, as well as transport of L-dopa over the gut barrier. This transwell assay represents a practical and essential step for validating intestinal absorption of bacterially produced L-Dopa.

            4. L-dopa production

The enzyme tyrosinase will be employed to convert L-tyrosine into L-dopa, which will subsequently be excreted into the zebrafish gut. We can quantify the L-dopa secretion by measuring the melanin that is created when L-dopa gets broken down.

Melanin synthesis pathway (Agarwal et al., 2018).

Modelling

When we do experiments, we don’t do them blindly. Before we start, we try to understand how the biological processes work and then try to turn that into a prediction. A simple example would be that bacteria need nutrients to grow, so we expect that if we give bacteria more nutrients then they will grow faster. This prediction is of course simple, but by using mathematics we can make more precise or difficult predictions. For example, if we give it twice the amount of nutrients, will it grow three times as fast, or maybe only one-and-a-half times as fast? After the experiment, we check to see if our model is correct by seeing if the prediction and the result we got are similar.

In our project we use modelling to predict the growth rate of bacteria in different environments, how much of the drug they can produce per hour and how to increase that productivity.

Human practices

Apart from the previously described labwork, we will also tackle our project in a societal context. In the iGEM competition, this is described as Human Practices (HP). Teams are motivated to investigate the needs of end users and stakeholders, and to adapt their project to these needs.

In our project’s HP, we set out to investigate the network of stakeholders involved in the path between our research and actual implementation of our project. Our goal is to analyse the wants and needs of these stakeholders, determine the roadblocks standing in the way of implementation, and direct our project to fit these needs.

Education

One of the weighing factors in societal implementation of a living biotherapeutical is the acceptance of the general public. We believe that education can help people understand the benefits and risks of a therapeutic like this, and thus help them make informed decisions regarding use of such a treatment.

We aim to teach the basics of synthetic biology to children and teenagers in schools, as well as the general public through YouTube videos.

Our team

Our project is part of the International Genetically Engineered Machine (iGEM) competition,  an annual global competition in which hundreds of teams go head to head in designing and developing a synthetic biology solution to a real-world challenge. We are a group of ten Bachelor’s and Master’s students from a diverse set of scientific backgrounds, and are tasked with tackling our problem both inside and outside the lab. All of this culminates in a final presentation to a large audience of scientists, investors, and industry leaders at the iGEM Grand Jamboree.