Line 21: | Line 21: | ||
<div class=“sec light_grey”> | <div class=“sec light_grey”> | ||
<div> | <div> | ||
+ | <div> | ||
<h3>Characterisation</h3> | <h3>Characterisation</h3> | ||
<h4>Nitric oxide sensor: NorV Promoter</h4> | <h4>Nitric oxide sensor: NorV Promoter</h4> | ||
Line 46: | Line 47: | ||
</div> | </div> | ||
</div> | </div> | ||
+ | </div> | ||
</div> | </div> | ||
<div class="sec white"> | <div class="sec white"> |
Revision as of 16:09, 19 October 2016
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.
Characterisation
Nitric oxide sensor: NorV Promoter
NorV promoter(PnorV, BBa_K2116002) 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]. We modeled the system based on parameters found in the literature. Our model suggested that endogenous production of NorR in E.coli [3] 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).
![](https://static.igem.org/mediawiki/parts/3/37/PnorVdoseresponse.png)
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].
AHL sensor: esabox/EsaR system
EsaR BBa_K2116001 is a transcriptional regulator of the Pantoea stewartii quorum sensing system [4] In the abscence of 3OC6HSL it can bind DNA and inhibit transcription. An esabox is a 18bp sequence where EsaR can bind. Unlike other quorum sensing regulators EsaR acts as a transcriptional repressor and not an activator.
In order to characterise represson efficiency of EsaR, we placed esaboxes after a constitutive promoter found on the registry (BBa_J23118).(Figure 3) During dose response tests we discovered that EsaR system displays non-sigmoidal dose response. With the intermediate AHL concentrations we tested, there's a dip in the dose response curve (Figure 3). This corresponds to concentrations close to the saturation point of the dose response. Our AHL sensor model could later explain the reason behind this behaviour. There is an interdependency between AHL concentration, EsaR concentration and number activated promoters. Which means there is a certain ratio of AHL:EsaR where we observe a shifted equilibrium towards the inactive state of the promoter.
Figure 3: Construct to test EsaR/esabox system. An esabox was placed after the constitutive promoter (BBa_J23118)..
Figure 4: Dose response curve for the EsaR/esabox test construct in Figure 3. At intermediate AHL (3OC6HSL) concentrations tested the dose response curve deviates from sigmoid behaviour. The reason behind this behaviour was later explained through our AHL sensor model.
Based on this insight from the model, we changed the amount of EsaR we express inside the cell to reduce the dip in the dose response curve. We tried putting two copies of EsaR under individual constitutive promoters( BBa_J23118; we tried expressing EsaR on a low copy plasmid instead of the initial medium copy plasmid; and we expressed EsaR under a stronger constitutive promoter (BBa_J23102)
AND gate
We generated AND gates by placing esaboxes either; i) as roadblocks after transcription start site of PnorV, preventing the polymerase from advancing or; ii) within the PnorV to establish competitive binding between NorR and EsaR or RNA polymerase and EsaR. (Check out our Part Collection)
Figure 5: One example of our AND gates responsive to NO and AHL. This is the AND gate we eventually used for our complete system. .
Since our complete system would express a recombinase under the control of this AND gate, we required one with low leakiness, and at least 4-fold activation. We characterised our collection and eventually decided to use the AND gate seen in Figure 5 since it showed the highest fold activation, and ideal leakiness for our purposes (Figure 6).
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.
- [4] Minogue, Timothy D. et al. "The Autoregulatory Role Of Esar, A Quorum-Sensing Regulator In Pantoea Stewartii Ssp. Stewartii: Evidence For A Repressor Function". Molecular Microbiology 44.6 (2002): 1625-1635.