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Team:Tuebingen/einweiterer

GLP.exe

GLP.exe

Abstract

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.

Project Inspiration

When committing to iGEM, we decided we firstly wanted to design a project which uses a new system of the field of synthetic biology, and secondly to make an impact with a product, which can actually be of use to the society. So when Dr. Pengfei Xia, one of our advisors proposed to design a new kill-switch system for bacteria, based on the Type I CRISPR system CRISPR/Cas3, we were determined to not only implement the system, but also find a way of making it a useful, universally applicable, tool. As Diabetes Mellitus Type 2 becomes more and more epidemic, with some of our team members relatives also suffering from it, and a problem for society and the healthcare system, we decided to focus our project on this disease.

We thought about changes in therapy, which would make it easier to comply with the therapy scheme and identified issues like the required daily injections and multiple drugs, which need to be taken at certain times during the day. Overall, we came to the conclusion that a probiotic bacterium synthesising a drug when required, and therefore lifting the burden of application from the patient, would be our way to go. On top of that, we realized that in 2017 Team AQA_Unesp had already tried to target Type 1 Diabetes Mellitus with a probiotic, hence we felt that the viability of the idea was supported by this [7]. Additionally, this approach would allow us to not only use our bacterium with the CRISPR/Cas3 system as biofactory, but also make it the therapeutic agent which is applied, making the biosafety function of CRISPR/Cas3, not only a tool, but also a requirement for safe application.

The idea of using GMOs as probiotics, is generally of great interest for chronic diseases. Therefore, we want to use the application in Type 2 Diabetes Mellitus as an example of the strengths and limitations of such a system, and use our public involvement to gather the society’s perception and opinion of such a therapeutic strategy. Overall, we consider our project to be an important trial for the use of synthetic biology in long-term therapy, during which the patient is not restricted to stay within a facility, but can live normally and without financial or other burdens of the disease. By designing a safe system which will prohibit the survival of GMOs within the environment, our project aims at enabling the use of probiotics in modern therapy.

Glucose-dependent Incretin secretion

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Our probiotic secretes Exenatid-4, an Incretin mimetic, in response to glucose availability in the human gut. The system works via the carbon catabolite repression system which will initiate the transcription of tetR. Upstream of our Exenatid-4 is a TetR repressible promoter, which ensures that Exenatid-4 is only transcribed in the presence of glucose. Furthermore, our Exenatid-4 is coupled to an N-terminal secretion tag and a C-terminal cell penetrating peptide (CPP), which together ensure the secretion and uptake of our drug increasing its bioavailability. Due to the immense variety of CPPs and the lack of quantitative information about their efficiency, we also developed a predictive software tool to allow for educated decisions on the design of CPPs.[LINK ZU INCRETIN SEITE]

CRISPR/Cas3 kill-switch

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.

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E.coli Nissle Characterisation

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.

Software

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.

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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.

References

  1. World Health Organization. (2016). Global Report on Diabetes. Available online; [Accessed 26.03.2019].
  2. Jens Juul Holst. The Physiology of Glucagon-like Peptide 1. (2007). Physiological Reviews. p.1409-1439.
  3. Lim, Gareth E., Brubaker, Patricia L. Glucagon-Like Peptide 1 Secretion by the L-Cell. (2006). 10.2337/db06-S020. Diabetes. p. S70-S77
  4. Copley, Kathrin & McCowen, Kevin & Hiles, Richard & L Nielsen, Loretta & Young, Andrew & Parkes, David. (2006). Investigation of Exenatide Elimination and Its In Vivo and In Vitro Degradation. Current drug metabolism. 7. 367-74. 10.2174/138920006776873490.
  5. Duan F, Curtis KL, March JC. Secretion of insulinotropic proteins by commensal bacteria: rewiring the gut to treat diabetes. Appl Environ Microbiol. (2008);74(23):7437–7438. doi:10.1128/AEM.01019-08
  6. Duan FF, Liu JH, March JC. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes. (2015);64(5):1794–1803. doi:10.2337/db14-0635
  7. iGEM Team AQA_Unesp
  8. Team NTU-Taida
  9. Sicard JF, Le Bihan G, Vogeleer P, Jacques M, Harel J. Interactions of Intestinal Bacteria with Components of the Intestinal Mucus. Front Cell Infect Microbiol. (2017);7:387. Published 2017 Sep 5. doi:10.3389/fcimb.2017.00387
  10. Barnhart MM, Lynem J, Chapman MR. GlcNAc-6P levels modulate the expression of Curli fibers by Escherichia coli. J Bacteriol. (2006);188(14):5212–5219. doi:10.1128/JB.00234-06
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  16. IDF (International Diabetes Federation. IDF Diabetes Atlas. (2017). Eighth Edition. Available from:
  17. Experimenta
  18. UN-SDG accessed: 01 May 2019 logos retrieved from here according to their guidelines
  19. Tuebingen:Human_Practices survey available under the link
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