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| − | <h2>Sensor Module</h2> | + | <h2>Title:</h2> |
| − | <h5><i>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.</i></h5> | + | <p>Paragraph content.</p> |
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| − | <p>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 <a href=“http://parts.igem.org/AHL”> AHL </a> as one example and constructed an AND gate that can detect NO and AHL. </p>
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| − | <p>Figure 1: AND gate design: Our AND gate operates through a nitric oxide sensing transcriptional activator (NorR) and an AHL sensitive repressor (EsaR).</p>
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| − | <h3>Characterisation</h3>
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| − | <h4>Nitric oxide sensor: NorV Promoter</h4>
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| − | <p>NorV promoter controls NO dependent transcription in E.coli <a href=“https://www.ncbi.nlm.nih.gov/pubmed/12529359”>[1]</a>. Gene expression can only be activated when NO binds the PnorV transcriptional activator NorR. </p>
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| − | <p> 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 <a href=“https://www.ncbi.nlm.nih.gov/pubmed/7996962”>[2].</a> We<a href=“https://2016.igem.org/Team:ETH_Zurich/Sensor_Module#nosensor”>modeled the system</a> based on parameters found in the literature. Our model suggested that endogenous production of NorR in <i>E.coli</i><a href=“https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2831303/”[3]</a> is enough for our desired range of sensitivity. Thus after successfully cloning the NorV promoter, we tested its activity with the endogenous NorR. <a href=“http://www.sigmaaldrich.com/catalog/product/sigma/d185?lang=en®ion=CH”>DETA/NO<a/> was used as a source of NO. Using our <a href=”https://2016.igem.org/Team:ETH_Zurich/NO_Release”>NO release model<a/> 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).
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| − | <p><b>Figure 2:</b> 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 <i>E.coli</i>. We can show that the activity range of the promoter is within the 20-200μM range that corresponds to inflammation in the gut<a href=“https://www.ncbi.nlm.nih.gov/pubmed/7996962”>[2].</a></p>
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| − | <h2>References</h2>
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| − | <li>[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.
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| − | <li>[2]Lundberg, J.O.N. et al. "Greatly Increased Luminal Nitric Oxide In Ulcerative Colitis". The Lancet 344.8938 (1994): 1673-1674.
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| − | <li>[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.
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Title:
Paragraph content.
Bigger title:
Second paragraph.
Thanks to the sponsors that supported our project:
