Motivation

IBD has recently becoma a major issue in devellopped European country. In the past few years, around 100 000 are newly diagnosed from Ulceric colitis and Crohn disease each year. In Europe about 1.4 milliom persons are concerned. In the United state The number of infected poeple reach 3 millions. Moreover in vivo diagnostic and test are extremely complicated to perform, while in vitro experiment are not reliable enough, because the intestine environement cannot be properly mimicated outside the human body. As the gut remain a black box like system, the causes of IBD still remain unknown, preventing proper cure. However, it is thougth that a disbalance in the gut microbiota, associated with some genetic factor migth be a possible cause of IBD. The current main issue is thus investigation in order to confirm and to establish which microbiota extending or shrinking can be associated with iBD.

Assumption

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

What dkind of circuit do we need we need to achieve our goal ? We need to associate two signals. A AND Gate seems to be the most appropriate circuit to be implemented in order to do that. However, as said previously, the gut behave as a black box. We need our bacteria to enter the gut, and we need to be able to know with which chemicals they interact once we separate them from the faeces. A simple AND Gate is here nott enough because any GFP fluorescent would have disappear in the timelaps between signal apparition and bacteria harvesting. We thus need our bacteria to 'remember' what happenned. To achieve this, we use a irreversible switch activated when both NO and AHL where present simultaneously. Then Un der the switch activation, GFP can be expressed. Later simply exposing the bacteria to AHL make them produce GFP, allowing us to know which microbiota was overactive when inflammation happened.

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 hexamric 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 inb 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 overpresent in some very heavy case of IBD, expecially in children. Thus it is also intersting yto investigate the role of Lactate in IBD occurences. 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 catalysist) are constitutively produced. in absence of Lacatte, LldR binds to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it binds to tyhe 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.

Switch Module

AND gate activation triggers Bxb1 production. Bxb1 is an invertase protein. 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.