Diabetes Mellitus is one of the main diseases causing premature death worldwide [16]. In 2014, 422 Million adults were diagnosed with Diabetes Mellitus worldwide [1], an alarmingly high number. This inspired us to look into alternative treatment strategies for Type 2 Diabetes Mellitus, as it marks the largest group of Diabetes patients. There already is a variety of treatment options, however they all require active participation of the patients and compliance to their individual therapy schemes. As compliance and persistence are key factors for therapeutic success, we, the iGEM Team Tübingen, want to revolutionize the treatment and its administration with the use of Synthetic Biology. We therefore developed GLP.exe a probiotic on the basis of Escherichia coli Nissle which secretes Exenatid, an incretin anologon, in response to glucose. To ensure its safe use as a GMO we invented a CRISPR/Cas3-based kill-switch integrated into our probiotic. This kill-switch is regulated by environmental factors and prevents the release and spread of our GMO into the environment. Our probiotic allows for a single-time application as it independently synthesises the drug when it is needed. To deepen our understanding of our probiotic strain we also worked on characterising E.coli Nissle via different means such as RNA-Seq and metabolic modelling. This characterisation will be benefitial for the iGEM and general scientific community in future projects. The use of GMOs, especially in the medical field, is a delicate topic and great efforts were made during our project to spread awareness and to educate the public about synthetic biology, GMOs and Diabetes.

In order to design an impactful project for this year’s iGEM season, we deemed it important to precisely understand and define the problem we wanted to work on.

Through extensive research into the factors impeding phage therapy, we identified the production process to be one of the most striking problems. In particular, the current methods are inefficient, lead to high impurities and contamination, require the cultivation of human pathogens in large quantities and causes regulatory problems due to imprecise manufacturing standards and a lack of adequate quality controls.

The implemented and newly developed CRISPR/Cas3 kill-switch allows for the safe use of our probiotic. Once the kill-switch is activated the Cas3 nuclease degrades the bacterial nucleic acid and therefore prevents the spread of GMOs into the environment. The kill switch is regulated by three environmental factors which are common in a healthy humans intestine: 37°C, availability of fatty acids in form of Acyl-CoA and N-Acetyl-Glucosamin (GlcNAc), released by the metabolism of mucus through commensal microorganisms [9]. As soon as the probiotic leaves its designated area, the repression of the kill switch is abrogated and the CRISPR/Cas3 system activated. The kill-switch therefore, is a concept that can be used for a diverse spectrum of therapies by exchanging the drug and/or the conditions.

Despite the wide use of E.coli Nissle (EcN) it is not characterised well enough. To enhance our knowledge of EcN and to provide crucial information to the scientific community as well as future iGEM teams, we investigated EcN’s transcriptome under different stress conditions. Understanding the complete transcriptome, the expressed genes, post-transcriptional modifications, single-nucleotide polymorphisms (SNPs) and additional properties of interest is essential for understanding genetic causes of adaptations to stress. The gained insight could lead to the development of more stress resistant strains, improving probiotic treatment to a large degree. Hence, we conducted large-scale RNA-Seq experiments for 12 conditions including aerobic and anaerobic environments. The conditions were chosen after a thorough evaluation of EcN’s growth under different strengths of the stress conditions. Additionally, we created the first ever metabolic model of EcN and made it available to the iGEM and general scientific community. Moreover, we modelled the interaction of EcN with three different bacterial communities - cutting-edge research and a novelty in the iGEM competition.

To transport Exendin-4 across the membrane of the enterocytes in the gut, we decided to utilize cell-penetrating peptides (CPPs) as a carrier. CPPs have already been proven to transport different cargos like insulin, from the gut to the bloodstream [98]. To better understand the mechanism of action of CPPs and to make an educated decision for choosing the CPP domain in our project design, we decided to generate a machine learning model to predict the cargo transport efficiency.

CPPs gain more and more attention in the scientific field, with multiple peptides being in clinical trials to deliver drug molecules to target sites in patients [99]. Since they can be used in various transport scenarios, many iGEM teams submitted CPPs to the registry in the past years. Therefore, we made our transport effectivity quantification software available to all future iGEM teams and the scientific community. To ensure excellent usability, we implemented a web GUI that allows multiple input formats.

Human Practices & Public Outreach

Since our project involves the use of GMOs, which are a constant topic of debate, we realised that we needed to invest into spreading awareness and especially education about GMOs, synthetic biology and Diabetes. We therefore contacted several experts and teamed up with a variety of institutions, researchers and also iGEM teams in order to address this. Several collaborations, meetups, exchanges and talks provided us with valuable information for our project and helped developing it over the year. Learn more about our Human Practices under Human Practices / Overview.

Wetlab Project Plan

To conclude, our projects required intensive cloning of multiple regulatory elements. For the parts, we used Biobricks that were sent with the 2019 shipping, Biobrick sequences from the database of iGEM, as well as new sequences, which are unique to our project, like the Exendin-4 fusion construct or the GlcNAc-6-P sensing system. After synthesis, the GOI constructs were amplified and cloned into vectors. These were used to transform competent E. coli cells that are commonly used for cloning applications, as they can be manipulated easily. Here, we can already test whether our GOI construct works and Exendin-4 is secreted in a glucose dependent manner.

At the same time the Cas3 system was isolated from E. coli K12, and the DNA with regulatory system was changed in E. coli K12 to our individual sensing mechanisms. The functionality of the kill switch and the different conditions for it can be tested already. If the construct shows the desired activity, it can be integrated into the E. coli Nissle 1917 genome. Finally, we hoped to combine our GOI with the Cas3 positive E. coli Nissle 1917 and test the functionality of our system as a whole. More precisely, we wanted to test if the generated strain secretes Exendin-4 in the presence of glucose, and if it destructs itself under conditions diverting from the gut.

In the future, we will proceed with experiments on human cell lines. Firstly, the transport of the Exendin-4 through a CaCo-2 cell monolayer will be tested to investigate, whether there will be a drug, which can reach the pancreas. Secondly, we will work with Rat Insulinoma cell line INS-1 cells, to examine whether Exendin-4 will induce an Insulin expression.

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