Team:ETH Zurich/Results

PAVLOV'S COLI

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Sensor Module

We successfully constructed and characterised several variants of a novel and modular AND gate. We also characterised the components separately and submitted them as new biobricks.

Our associative learning system requires simultaneous detection of two signals. The first signal is nitric oxide, which serves as a marker for inflammation. The second signal can be any number of different microbiome markers inside the gut. We chose AHL as one example and constructed an AND gate that can detect NO and AHL.

Figure 1: AND gate design: Our AND gate operates through a nitric oxide sensing transcriptional activator (NorR) and an AHL sensitive repressor (EsaR).

Characterisation

Nitric oxide sensor: NorV Promoter

NorV promoter controls NO dependent transcription in E.coli [1]. Gene expression can only be activated when NO binds the PnorV transcriptional activator NorR.

Initially we wanted to see how much NorR we would require in order to sense a range of NO from 20-200μM, which is the range that is typically seen in IBD [2]. Wemodeled the system based on parameters found in the literature. Our model suggested that endogenous production of NorR in E.coli is enough for our desired range of sensitivity. Thus after successfully cloning the NorV promoter, we tested its activity with the endogenous NorR. DETA/NO was used as a source of NO. Using our NO release model for DETA/NO, we could show that the PnorV promoter is active within the range of 20-200μM of NO we would like to detect in the gut (Figure 2).

Figure 2: PnorV dose response curve for a range of DETA/NO concentrations that corresponds to 7-70μM of NO. Based on the insights we got from our model, we tested the promoter only in presence of endogenous NorR in E.coli. We can show that the activity range of the promoter is within the 20-200μM range that corresponds to inflammation in the gut[2].

References

• [1]Gardner, A. M. "Regulation Of The Nitric Oxide Reduction Operon (Norrvw) In Escherichia Coli. ROLE OF Norr AND Sigma 54 IN THE NITRIC OXIDE STRESS RESPONSE". Journal of Biological Chemistry 278.12 (2003): 10081-10086. Web. 16 Oct. 2016.
• [2]Lundberg, J.O.N. et al. "Greatly Increased Luminal Nitric Oxide In Ulcerative Colitis". The Lancet 344.8938 (1994): 1673-1674.
• [3] Tucker, N. P. et al. "Essential Roles Of Three Enhancer Sites In  54-Dependent Transcription By The Nitric Oxide Sensing Regulatory Protein Norr". Nucleic Acids Research 38.4 (2009): 1182-1194.

Thanks to the sponsors that supported our project:

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