DESIGN

OVERVIEW

Inflammatory bowel disease (IBD) is an insufficiently understood chronic disease, whose increasing incidence, lack of proper treatment and diagnostic methods make it an urgent problem to solve^[1]. The current methods for diagnosing IBD mainly rely on endoscopies, which are invasive and do not allow to properly characterize the microbiome of patients, believed to be implicated in pathogenesis.

We designed an alternative solution for in vivo diagnosis, using hydrogel-encapsulated Pavlov's coli. In order for Pavlov's coli to efficiently gather information on important aspects of IBD, such as inflammation and microbiome composition, we designed our system such that it consists of:

A biological AND gate that allows our system to associate known IBD markers present in the gut.
A switch acting as memory, which is irreversibly flipped from "OFF" to "ON" if both markers are simultaneously detected by the AND gate. This switching mechanism renders a reporter (GFP, see later) sensitive to one of the two biomarkers previously encountered by our logic AND gate.
Information sensed by the AND gate is stored (remembered) by the reporter via the switching mechanism and can be read-out later in a laboratory. This conditional activation of our reporter justifies the name of our diagnostic tool - Pavlov's coli.

The encapsulation should allow for diffusion of molecules, while preventing our bacteria from colonizing the gut, therefore providing a non-invasive tool, which does not disrupt the microbiome.

Fig. 1: Structure of Pavlov's coli's genetic circuit. (1) The sensor module comprises an AND gate which associates an inflammation marker with a candidate marker, representative of microbiome composition. (2) The switch module consists of an irreversible genetic switch. (3) The reporter module can be used to retrieve information about the biomarkers sensed by our AND gate.

An additional feature of our system is that it allows for multiplexing, meaning processing multiple signals through one channel: in our system, the reporter module (3) is activated by one of the two markers also required to activate the sensor module, i.e. the AND gate (1) (see Fig. 1). The architecture of our system has the following implication: a suite of bacterial populations, each responsive to different IBD-relevant candidate markers (e.g. different AHL molecules, lactate, etc.) as well as to an inflammation marker (e.g. NO), can be engineered, complexed together and administered to a patient; upon recovery, re-exposure of faecal samples to a previously encountered biomarker can be read out through our reporter module (see Fig. 2).

Fig. 2:Mode of action of Pavlov's coli. Step 1: After administration of our bacteria, biological markers can be sensed in the gut through a logical AND gate; the event is memorized through a switching mechanism. Step 2: Following excretion and retrieval of our bacteria, the memory can be read-out by exposing the samples containing our bacteria to a host of IBD-relevant candidate markers.

Therefore, Pavlov's coli holds promise as a non-invasive, modular, multipleable, in vivo diagnostic tool for IBD investigation.

Sensing in the gut

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that remain reliable. We designed our circuit as interchangeable, easily tunable parts, each of which is backed by a computer model and experimental data.

IBD is normally accompanied by severe inflammation of the intestine. Several sources in the literature report on a sharp increase in Nitric Oxide (NO) levels in inflamed areas. Based on this, we chose to incorporate a sensor for NO as part of a logic AND gate in our engineered bacteria^[1].

Along with the presence of NO, Pavlov's coli's logic gate requires a second input in order to activate an irreversible switch (see Fig. 1; also, see section below). As the second input, we chose a marker which can provide information on microbiome composition: AHL. A number of AHLs have been found to be informative as to specific functions (e.g. biofilm formation) and microbiome composition^[2].

In an alternative design, we chose to replace the AHL sensor with lactate sensor. In an interview with an expert on IBD, Prof. Lacroix, we discussed the potential of bacterial metabolites as an alternative microbiome marker. One such a candidate turned out to be lactate, which is produced and metabolized by different bacteria in the gut. Although it is not as informative as AHL to the end of microbiome characterization, there is a strong interest in developing methods for lactate detection in the gut. Due to its nature as an intermediate metabolite, it is currently not possible to measure lactate concentrations in fecal samples of patients, adding appeal to the development of Pavlov's coli.

DETAILED IMPLEMENTATION

Our system is composed of a logic AND gate, a switch and a reporter, behaving together as an associative learning circuit.

The AND gate is composed of the P_norV promoter and an operator. The promoter can be activated by NorR upon its binding with NO. In the absence of NO NorR behaves as a repressor. The operator is either an esabox, for AHL sensing, or LldO, for lactate sensing. These operators are repressed by EsaR and LldR respectively and repression is relieved upon binding of AHL or lactate respectively (see Fig. 3). The modular nature of our system allowed us to easily exchange the esabox with LldO, through one site-directed mutagenesis.

The switch module, under the control of the AND gate, is responsible for flipping a reporter cassette irreversibly. We have designed three different switches, based on integrases^[3], CRISPR/Cas9^[4] and a novel genetic switch based on the recently discovered CRISPR/Cpf1^[5]

The reporter module can drive expression of two different fluorescent proteins. In its default OFF state, i.e. prior to AND gate activation, the module expresses the orange fluorescent protein mNectarine under control of an operator (esabox or LldO). Upon successful flipping, activation of the operator results in GFP expression (see Fig. 3). This mechanism is what makes multiplexing possible. Note that several reporter plasmids are present in the cell and the fraction of flipped cassettes can give us quantitative information on the signal intensity of the inputs activating the AND gate (link to switch modeling page).

We chose E. coli as a host organism because it is easy to manipulate and engineer. In future the system could be transferred into a Lactobacillus acidophilus or Lactococcus lactis host, which are regarded as safe to ingest, this was recommended to us by Prof. Gerhard Rogler.

Fig. 3:Structure of our genetic circuit for IBD diagnosis. Note how NO and AHL are both necessary to express a molecular switch (here: Bxb1) which in turn is responsible for flipping a reporter cassette to its ON state, i.e. GFP can be expressed upon induction with AHL. Note that failure to activate the AND gate would result in mNectarine expression upon induction with AHL.

CONCLUSION

Our design for Pavlov's coli creates a novel investigation tool for IBD that:

Does not disrupt the gut microbiome
Can associate potential new markers of IBD pathogenesis to an established marker of IBD and memorize the event
Allows for multiplexing
Is flexible and easily adjustable to new potential markers due to its modular nature.

REFERENCES

[1] G. Kolios, V. Valatas, S. G. Ward, Immunology 2004, 113, 427-437.
[2] M. Schuster, D. J. Sexton, S. P.
Diggle, E. P. Greenberg, Annu Rev Microbiol 2013, 67, 43-63.
[3] J. Bonnet, P. Subsoontorn, D. Endy, Proc
Natl Acad Sci U S A 2012, 109, 8884-8889.
[4] H. Huang, C. Chai, N. Li, P. Rowe, N. P. Minton, S. Yang, W.
Jiang, Y. Gu, ACS Synth Biol 2016.
[5] B. Zetsche, J. S. Gootenberg, O. O. Abudayyeh, I. M. Slaymaker, K.
S. Makarova, P. Essletzbichler, S. E. Volz, J. Joung, J. van der Oost, A. Regev, E. V. Koonin, F. Zhang, Cell 2015, 163, 759-771.