Team:ETH Zurich/Description
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Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) describes the chronic inflammations of parts of the intestine and is a collective of several further specified illnesses. The most common conditions are ulcerative colitis and Crohn's disease. It is classified as an autoimmune disease for which no cure has been developed. Current treatments include immunosuppression, surgery, antibiotics and nutritional therapies. This disease is a severe burden for the patients as well as it causes increasing direct (treatment) and indirect (absenteeism from work) costs for society. Furthermore, only in Europe 2.5 million people are affected by IBD and the number of cases is increasing world-wide1.
Unfortunately there are no characteristic blood markers to distinguish the different forms of IBD. The diagnosis relies mostly on the location of inflammation observed during a colonoscopy. Also the underlying trigger of the disease is not completely understood but correlation studies proposed factors such as diet, genetic predisposition, breach of the intestinal barrier and unfavorable alteration of the microbiota, called dysbiosis2. The diversity of the microbiota is noticeably reduced2,2 in IBD patients and the composition of the gut flora changes from symbiotic to predominantly pathobiotic microbes1.
The inflammation of the intestine partially interrupts the integrity of the layer of epithelial cells lining the intestine. This cell layer separates the gut lumen containing trillions of microbes from the body. The damage to this essential barrier compromises the selectivity and allows for penetration of immunogenic antigens from the lumen across the epithelial layer1, which enhances the inflammation reaction.
Sensing of Markers for IBD
Nitric Oxide
Beside the penetration of immunogenic antigens across the epithelial layer, there is also non-normal leakage of inflammation markers into the gut lumen. One of these molecules is nitric oxide (NO•, t1/2 < 6 seconds1) and is one of the molecules we are going to sense with our system. The sensing of NO• with E. coli has already been described by Archer et al.1 in 2012. This work provides us with the relevant genetic elements and helps us to design this system for our purpose. Additionally, they present their system as a rapid detection system for IBD related disease flare-ups which would allow for an immediate intervention.
NorR is capable of binding NO• with its mononuclear non-heme iron center. While other sensor proteins are not only specific for NO• but also for other NOs species, NorR binds specifically the NO• radical. NorR is constitutively bound as a hexamer upstream of the norVW promoter but inhibiting transcription in absence of NO•. Once NO• binds to NorR, its ATPase activity is triggered and provides energy to form a productive interaction with the σ54 - RNA polymerase holoenzyme1.
N-Acyl Homoserine Lactones
In addition to a general inflammation marker we want to sense molecules secreted by the microbiota in order to identify the bacteria. One well-known class of molecules secreted by many bacterial species belongs to the quorum sensing (QS) system. QS molecules act as bacterial hormones among and between species which control for example the formation of biofilms and growth behaviour. Furthermore, QS molecules can alter the microbiota's composition1. The best known subclass of QS molecules are the N-acyl homoserine lactones (AHL) which will be identified by our living biosensor.
One of the AHLs to be found upregulated in IBD1 is 3-hydroxy-hexanoyl-HSL (3-OH-C6-HSL). A well characterized regulatory protein that senses a very similar HSL (3-oxo-C6-HSL) is EsaR. The special feature of EsaR is its regulatory behaviour: while most HSL-responsive elements are inducible activators, EsaR is a repressor that dissociates from the DNA in presence of HSL. This is important for our circuit as a repressor is thought to be less leaky than an activator.
As our target HSL is not the natural ligand for EsaR, we applied a directed evolution strategy to change its specificity.
Associative Learning Circuit
Overview
To serve as a diagnostics and research tool, our system should not only be able to sense a single molecule alone but should associate an inflammation marker - in our case NO• - with a potential trigger of the inflammation itself. Thus, we implemented an associative learning circuit that allows for the detection of the temporal and spatial presence of two markers.
Nitric oxide and 3-OH-C6-HSL are only two possbile markers of IBD. There exist many more that are definitively worth to be further investigated and are ideally observed in parallel. This is why we extended the AND-gate by a learning component. While the number of distinguistable reporters (e.g. fluorophores) is limited, our system allows for simultanious observation of a multitude of parallel measured markers. Our Pavlov's Coli learn the occurence of the presence of two markers and store this information in their DNA until readout.
We designed our system in a way that allows fast and easy demultiplexing of a complex mixture of different reporter strains. If the reporter strains encounter again the with inflammation associated marker, they generate an easily observable output: fluorescence. This was achieved by integrating a second AND-gate that relies on the successful learning process.
Overview
References:
Thanks to the sponsors that supported our project:
