Difference between revisions of "Team:ETH Zurich/Results"

Line 173: Line 173:
 
<div class="sec white">
 
<div class="sec white">
 
<div>
 
<div>
−
<h2>SWITCH and REPORTER MODULES</h2>
+
<h2>SWITCH and REPORTER MODULES</h2>
−
<p>The switch and the reporter together form the memory and read-out part of our associative learning system.</p> Our reporter is a dual fluorescent construct, expressing the red fluorescent protein
+
<p>The switch and the reporter together form the memory and read-out part of our associative learning system.</p> Our reporter
−
mNectarine when the system is OFF, and GFP when the memory has been triggered. This switching is induced by a recombinase that can flip a directional promoter <a href=>J23118</a>  
+
is a dual fluorescent construct, expressing the red fluorescent protein mNectarine when the system is OFF, and GFP when
 +
the memory has been triggered. This switching is induced by a recombinase that can flip a directional promoter <a href=>J23118</a>
 
</div>
 
</div>
 
</div>
 
</div>
Line 202: Line 203:
 
</div>
 
</div>
 
</div>
 
</div>
 +
</div>
 +
</div>
  
 
</html>
 
</html>
 
{{:Template:ETH_Zurich/footer}}
 
{{:Template:ETH_Zurich/footer}}

Revision as of 23:56, 19 October 2016

PAVLOV'S COLI

ACHIEVEMENTS

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

  • SENSOR MODULE

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

    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 a 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).

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]. The dose response does not reach full saturation since too much nitric oxide effects growth of bacteria.Measured 6 hours after induction. Error bars represent S.D. from three technical replicates.

We also wanted to demonstrate that we are indeed using the endogenous NorR to activate our cloned promoter. For this purpose we used the Keio norR KO strain and compared it to its WT parent strain and proved that the endogenous NorR is activating our system.

Figure 3:A norR knock out E.colistrain was used to demonstrate that endogenous NorR activates PnorV in abscence of exogenously expressed protein. Induced with 5000μM DETA/NO and measured 6 hours after induction. Error bars represent S.D. from three technical replicates.

AHL sensor: esabox/EsaR system

Figure 4: Mechanism of transcriptional repression by EsaR. In the abscence of 3OC6HSL EsaR dimerizes and binds the esabox sequence to repress transcription.

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 (Figure 4). 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.

Figure 5: Construct to test EsaR/esabox system. An esabox was placed after the constitutive promoter (BBa_J23118)..

In order to characterise represson efficiency of EsaR, we placed esaboxes after a constitutive promoter (BBa_J23118).(Figure 5) 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 6). 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.

Figure 6: Dose response curve for the EsaR/esabox test construct in Figure 5. 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. Measured 3 hours after induction. Error bars indicate S.D. of three technical replicates

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. Our strategy was to put two copies of EsaR under individual constitutive promoters( BBa_J23118; expressing EsaR on a low copy plasmid instead of the initial medium copy plasmid; and expressing EsaR under a stronger constitutive promoter (BBa_J23102). In the end we obtained the best results with increasing EsaR expression through the stronger constitutive promoter (BBa_J23102). Even though we cannot obtain fully sigmoidal behaviour, we managed to reduce the effect of this phenomenon on the EsaR dose response. (Compare Figure 5 and 6). Consequently we decided to use this construct for assembling our complete system.

Figure 6: Esar dose response for expression under strong constitutive promoter (BBa_J23118). The dose response behaviour is still not sigmoidal, however the effect of the dip seen in Figure 5 is significantly attenuated by increasing EsaR expression. Measured 3 hours after induction. Error bars indicate S.D. of three technical replicates.

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 for examples)

Figure 8: One example of our AND gates responsive to NO and AHL. This is the AND gate we eventually used for our complete system. Nomenclature: NRB-NorR binding site, sigma54B-RNA polymerase binding site.

Our complete system would express a recombinase under the control of this AND gate. Our model for the switch worked best when we assumed low leakiness. Thus after we characterised our collection we decided to use the AND gate seen in Figure 5. It showed the highest fold activation, thus the lowest leakiness(Figure 9). MATTIA, A FEW WORDS AND A PLOT ABOUT FOLD-ACTIVATION BEING MOST IMPORTANT?.

Figure 9: The AND gate we selected to use for our complete system displays 4-fold activation and low leakiness, fulfilling the criteria suggested by our model for the switch Induction was with 5mM of DETA/NO Measured 3 hours after induction. Error bars indicate S.D. of three technical replicates.

An important requirement for our AND gate is that the second input should be easily exchangeable. This is where our learning circuit gets its flexibility from. This was an important motivation behind choosing an activator system for the NO part, and a repressor system for the AHL part of our AND gate. In order to demonstrate this flexibility, we exchanged the esaboxes with the lldO operators, allowing us to create an AND gate responsive to lactate and NO (Figure 7).

Figure 9: An alternative AND gate responsive to NO and lactate. Demonstrates the flexibility of our system.

SWITCH and REPORTER MODULES

The switch and the reporter together form the memory and read-out part of our associative learning system.

Our reporter is a dual fluorescent construct, expressing the red fluorescent protein mNectarine when the system is OFF, and GFP when the memory has been triggered. This switching is induced by a recombinase that can flip a directional promoter J23118

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.

Thanks to the sponsors that supported our project: