Revision as of 21:26, 19 October 2016

DESIGN

OVERVIEW

Inflammatory bowel disease (IBD) is an insufficiently understood world-wide problem, and only invasive diagnostic tools such as colonoscopy or biopsy are currently available.

We designed an alternative solution for in vivo analysis, using hydrogel-encapsulated E. coli, containing (i) a biological AND gate which allows our system to associate two disease markers present in the gut, (ii) a switch acting as memory, which is flipped from 0 to 1 if both markers are detected, and (iii) a reporter to be readout later in a laboratory, providing information about the detected biomarkers.

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that are reliably reproducible. We designed our circuit as interchangeable, easily tuneable parts. This allows multiplexing, one of our circuit’s major advantages.

Therefore, Pavlov's coli is the best solution for a non-invasive, modular, multiplexing, in vivo tool to investigate IBD’s unknown causes.

Figure 1: Schematic view of the model structure.

REQUIREMENT

In order to carry this investigation, we need a factor specific to inflammation. Here, we choose Nitric Oxide, which is present in the gut when there are lesion and inflammation. We also need a proper microbiota marker. Each microbiota naturally produces specific AHL. We thus chose AHL as a microbiota specific marker. Finally, we also need a reporter. We chose Mnectarine a redish fluorescent protein, that will be expressed in case of absence of associated inflammation with the microbiota, and GFP expressed in case a match between one specific microbiota and inflammation is found.

SYSTEM

As stated before, current diagnosis tools in IBD involve invasive solution such as colonoscopy and biopsy. And because of the lack of information regarding IBD causes, and the lack of reliability when analysing samples in vitro, research stagnates. Therefore an adaptive learning tool for in vivo diagnostic and investigation was designed. Our system is composed of a logic AND gate, a switch and a second AND gate. The switch acts as a memory bit that can be flipped from 0 to 1 when it detects both inflammation and a specific microbiota disbalance through the AND gate. We currently developed three different possible switches, based on integrase kinetic, CRISP/Cas9 and the recently discovered CRISP/Cpf1 complex. Our first AND gate sensor is sensitive to both microbiota specific AHL, and inflammation marker NO. A later version allow us to sense lactate, as recent studies tends to demonstrate its role in heavy case of IBD in children. For minimal system disruption, we chose E.Coli which is native to gut microbiota. A future improvement would be to transfer this circuit into a Lactobacillus since they are also native and non pathogenic.

SYSTEM OVERVIEW

Figure 2: System Overview

DESIGN MOTIVATION

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that are reliably reproducible.We designed our circuit to be as modular as possible. As the interest of this genetic circuit mainly lays when multiplexing is possible, we designed it such that you can associate any candidate signal just by changing the promoter. Thus, constructing a simple library of AHL, you can create a library of E. Coli capable to sense a wide range of microbiota signals. Moreover, our model shows that the system is easily tunable playing with NorR, Esar, and integrase production and degradation rates to fit to the required range of detection, and thus would be the best solution for a modular multiplexing investigation tool.

INPUTS SENSOR

We need a NO sensor and a AHL sensor. Later we will also need a Lactate sensor, as recent researches tend to asses that lactate is present in extremely high quantity in some heavy case of IBD in children. Our input sensor is composed of a PnorV promoter associated with esaboxes situated downstream the promoter and upstream the reporter gene. In an improved version of this sensor, the esabox (to which EsaR can bind and act as a roadblock , preventing gene transcription) will be put in different place around the PnorV promoter. The idea is to see if competitive binding can bring better result than traditional independent gene activation and inhibition. More over it is known that PnorV activation mechanism under NO/NorR binding involves DNA looping aroung the promoter. As a consequence, a low efficiency of EsaR road block behavior is expected as the looping could prevent it from binding to the esaboxes.

SWITCH

We currently developed three different possible switches, based on integrase kinetic, CRISP/Cas9 and the recently discovered CRISP/Cpf1 complex. When both NO and AHL are present the hybrid promoter is activated and lead to intregrase protein production. Intregrase is a protein that is capable of binding to a particular DNA sequence referred as AttP and AttD. After binding the DNA sequence is cut and inverted. The switch acts here as a memory bit that can be flipped from 0 to 1 in an irreversible way. The flipped sequence contains a constitutive promoter associated with esaboxes, and thus negatively regulated by EsaR.

REPORTER

On each side of the flipped sequence are placed a mNectarine and a GFP gene respectively. Depending of the flipping state of the DNA sequence, the cell produces either mNectarine of GFP. As explained above this gene expression is regulated by ,EsaR. Thus once the DNA is switched, the cell produces GFP if AHL is present in the medium.

GENETIC CIRCUIT

Figure 3: Genetic Circuit

NO SENSOR

We use here the PnorV promoter, specific to NO sensing. We also use a constitutively produced NorR protein. NorR exist under a dimer form in the cell. Each dimer is able to bind to the 3 binding site present on PnorV. Then those three dimer assemble in a hexameric ring like structure. When NO is present in the medium, it binds to the structure and activate the promoter.

AHL SENSOR

In addition to the PnorV promoter, we had downstream esaboxes. Esar is constituvely produced in our bacteria. Just like NorR it exists as a dimer in the cell. The dimer form binds to the esaboxes forming a roadblock and unabling gene transcription when NO only is present. When AHL enter the cell it binds to the EsaR dimer and free the promoter, allowing transcription.

LACTATE SENSOR

Recent studies highlighted the fact that Lactate seems to be over-present in some very heavy case of IBD, especially in children. Thus it is also interesting to investigate the role of Lactate in IBD occurrences. We uses here a modified version of the Plac promoter. two LldR binding sites O1 and O2 are situated upstream the promoter. LldR and LldD (Lactate -> Pyruvate catalyst) are constitutively produced. in absence of Lactate, LldR binds to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it binds to the LldR dimer and free the promoter. We introduced LldD to increase the threshold of Lactate sensing.

AND GATE

The AND gate is the association of both NO sensor and AHL or Lactate sensor. It is constituted by a hybrid promoter composed of the PnorV promoter and the downstream esaboxes. Model shows that playing with NorR and EsaR production and degradation rates can allow a fine tuning of the sensor to adapt to the wished detection range.

SWITCH MODULE

AND gate activation triggers integrase production. Its role is to inverse the DNA strand containing the GFP gene.

REPORTER MODULE

the reporter module is just constituted of some esaboxes and the GFP gene. Under AHL presence, the reporter (GFP) is expressed.

CONCLUSION

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that are reliably reproducible. We designed our circuit as interchangeable, easily tuneable parts. This allows multiplexing, one of our circuit’s major advantages for a non-invasive, modular, multiplexing, in vivo tool to investigate IBD’s unknown causes.

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