Team:ETH Zurich/Description

PAVLOV'S COLI

Pavlov’s Coli: An associative learning based diagnostics tool applied to IBD

The ETH Zurich iGEM team 2016 will focus on the diagnosis monitoring of inflammatory bowel disease. Current diagnostic methods are invasive and rely on biomarkers that are not sufficiently disease-specific. We have engineered E. coli to detect several disease-specific biomarkers, memorize this event, and allow specific readout of the memory state. While the sensor cells travel through the gut, simultaneously occurring signals are memorized by activating an AND gate which triggers a recombination-based unidirectional switch and commits the observation to memory. After isolation from the patient’s faeces, the memory can be read out through the expression of a fluorescent protein induced by the addition of the candidate biomarker. Thus a single fluorescent protein can differentiate between many different candidate markers. A community of sensor cells can be utilized at the same time, enabling a high degree of multiplexing. Pavlov’s Coli is a non-invasive diagnostic tool for a large selection of specific biomarkers associated with IBD.
Furthermore, we use directed evolution to adapt a natural repressor of Erwinia stewartii for sensing a disease-specific AHL with our genetic circuit.

Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) describes the chronic inflammations of parts of the intestine and is a collective of several further specified illnesses. The most common conditions are ulcerative colitis and Crohn's disease. It is classified as an autoimmune disease for which no cure has been developed. Current treatments include immunosuppression, surgery, antibiotics and nutritional therapies. This disease is a severe burden for the patients as well as it causes increasing direct (treatment) and indirect (absenteeism from work) costs for society. Furthermore, only in Europe 2.5 million people are affected by IBD and the number of cases is increasing world-wide1.
Unfortunately there are no characteristic blood markers to distinguish the different forms of IBD. The diagnosis relies mostly on the location of inflammation observed during a colonoscopy. Also the underlying trigger of the disease is not completely understood but correlation studies proposed factors such as diet, genetic predisposition, breach of the intestinal barrier and unfavorable alteration of the microbiota, called dysbiosis2. The diversity of the microbiota is noticeably reduced3,4 in IBD patients and the composition of the gut flora changes from symbiotic to predominantly pathobiotic microbes5.
The inflammation of the intestine partially interrupts the integrity of the layer of epithelial cells lining the intestine. This cell layer separates the gut lumen containing trillions of microbes from the body. The damage to this essential barrier compromises the selectivity and allows for penetration of immunogenic antigens from the lumen across the epithelial layer6,7 which enhances the inflammation reaction.

Sensing of Markers for IBD

Nitric Oxide

Figure 1: NorR constitutively binds to DNA. Only when NO• is present it activates the transcription of the gene under control of the norVW promoter (adapted from Green et al.)

Beside the penetration of immunogenic antigens across the epithelial layer, there is also non-normal leakage of inflammation markers into the gut lumen. One of these molecules is nitric oxide (NO•, t1/2 < 6 seconds7) and is one of the molecules we are going to sense with our system. The sensing of NO• with E. coli has already been described by Archer et al.8 in 2012. This work provides us with the relevant genetic elements and helps us to design this system for our purpose. Additionally, they present their system as a rapid detection system for IBD related disease flare-ups which would allow for an immediate intervention.
NorR is capable of binding NO• with its mononuclear non-heme iron center. While other sensor proteins are not only specific for NO• but also for other NOx species, NorR binds specifically the NO• radical. NorR is constitutively bound as a hexamer upstream of the norVW promoter but inhibiting transcription in absence of NO•. Once NO• binds to NorR, its ATPase activity is triggered and provides energy to form a productive interaction with the σ54 - RNA polymerase holoenzyme9.

N-Acyl Homoserine Lactones

Figure 2: DNA- and 3-oxo-C6-HSL bound, dimeric form of TraR, a close homolog of EsaR (PDB: 1L3L, edited with UCSF Chimera1)

In addition to a general inflammation marker we want to sense molecules secreted by the microbiota in order to identify the bacteria. One well-known class of molecules secreted by many bacterial species belongs to the quorum sensing (QS) system. QS molecules act as bacterial hormones among and between species which control for example the formation of biofilms and growth behaviour. Furthermore, QS molecules can alter the microbiota's composition1. The best known subclass of QS molecules are the N-acyl homoserine lactones (AHL) which will be identified by our living biosensor.
One of the AHLs to be found upregulated in IBD1 is 3-hydroxy-hexanoyl-HSL (3-OH-C6-HSL). A well characterized regulatory protein that senses a very similar HSL (3-oxo-C6-HSL) is EsaR from Erwinia stewartii that was used by a previous iGEM team. The special feature of EsaR is its regulatory behaviour: while most HSL-responsive elements are inducible activators, EsaR is a repressor that dissociates from the DNA in presence of HSL. This is important for our circuit as a repressor is thought to be less leaky than an activator.
As our target HSL is not the natural ligand for EsaR, we applied a directed evolution strategy to change its specificity.

Associative Learning Circuit

Overview

To serve as a diagnostics and research tool, our system should not only be able to sense a single molecule alone but should associate an inflammation marker - in our case NO• - with a potential trigger of the inflammation itself. Thus, we implemented an associative learning circuit that allows for the detection of the temporal and spatial presence of two markers.
Nitric oxide and 3-OH-C6-HSL are only two possbile markers of IBD. There exist many more that are definitively worth to be further investigated and are ideally observed in parallel. This is why we extended the AND-gate by a learning component. While the number of distinguistable reporters (e.g. fluorophores) is limited, our system allows for simultanious observation of a multitude of parallel measured markers. Our Pavlov's Coli learn the occurence of the presence of two markers and store this information in their DNA until readout.
We designed our system in a way that allows fast and easy demultiplexing of a complex mixture of different reporter strains. If the reporter strains encounter again the with inflammation associated marker, they generate an easily observable output: fluorescence. This was achieved by integrating a second AND-gate that relies on the successful learning process.

Biological Implementation: Recombinase

Sensor AND-gate:


At an inflammation spot, nitric oxide activates NorR and triggers the transcription of the bxb1 integrase gene. The transcription can only proceed if 3-OH-C6-HSL is present. The HSL lets the repressor EsaR dissociate from the regulatory element (esaBox) on the DNA and thus annihiliates its roadblock activity.

Learning:


Once the bxb1 gene is successfully transcribed and translated, Bxb1 binds to the attP and attB recombination sites flanking a constitutive promoter and inverts it. As attP and attB are destroyed through inversion, Bxb1 mediated recombination acts as a one-way switch.

Reporter AND-gate:


The constitutive promoter, now being placed upstream of the reporter protein GFP, is further under control of another esaBox, the binding site of EsaR.
After the system now has learnt to respond to the associated stimulus alone, the expression of GFP can easily be induced by just exposing it to the stimulus again, e.g. EsaR's ligand.

Biological Implementation: CRISPR/Cpf1

An alternative to a recombinase-based switch is the usage of the 2015 characterised CRISPR/Cpf1 system. Instead of cutting both DNA strands at the same position, Cpf1 cuts the DNA with an offset of four or five nucleotides, thus producing single-stranded overhangs. It is suggested that this is advantageous for genome editing via non-homologous end-joining.
We will use the features of Cpf1 to create an AND-gate controlled one-way switch with finally the same functionality as the recombinase-based switch.
For this, a reporter construct is stably integrated into the genome of E. coli whereas Cpf1 and its guide RNAs will be expressed from a plasmid.

Sensor AND-gate:


At an inflammation spot, nitric oxide activates NorR and triggers the transcription of Cfp1. The transcription can only proceed if 3-OH-C6-HSL is present. The HSL lets the repressor EsaR dissociate from the regulatory element (esaBox) on the DNA and thus annihiliates its roadblock activity. The guide RNAs are expressed constitutively at a high level.

Learning:


Once Cfp1 is expressed, it is brought to the cutting-sites by the two distinct guide RNAs. There, Cfp1 cuts out the mNectarine gene while creating sticky ends. These will then be ligated by endogeneous ligases by NHEJ which reconstitutes the GFP gene.

Reporter AND-gate:


The reconstituted GFP gene is now under the control of a constitutive promoter regulated be an esaBox, the binding site of EsaR.
After the system now has learnt to respond to the associated stimulus alone, the expression of GFP can easily be induced by just exposing it to the stimulus again, e.g. EsaR's ligand.

Directed Evolution of EsaR

In order to change EsaR's specificity towards an IBD related HSL, we need to apply directed evolution on this repressor protein. The variant of EsaR that we used was already an improved version with a D91G mutation that has an increased signal sensitive compared to the wildtypX. We combine several approaches to find new variants of EsaR that are responsive to our target. To select for these variants we have different constructs that form a dual selection system. This systems allows for negative selection ("killing") of variants that still react to the former HSL (3-oxo-C6-HSL) and positive selection ("survival") of variants that respond to the new target HSL (3-OH-C6-HSL). It consists of a fusion protein that is composed of an antibiotic resistance and an enzyme that converts a non-toxic compound into a cellular toxin. We test the combination of the chloramphenicol acetyltransferase (CAT) and the uracil phosphoribosyltransferase (UPRT)1 as well as the herpes simplex virus thymidine kinase (hsvTK) fused to the aminoglycoside phosphotransferase (APH)1. Whereas CAT and APH confer resistance for the positive selection step, UPRT and hsvTK are necessary for the negative selection.
UPRT normally converts uracil into uridine monophosphate (dUMP). 5-fluorouracil is metabolised by UPRT to 5-fluoro-dUMP which irreversibly blocks the thymidylate synthase (thyA), a key enzyme for the production of pyrimidine nucleosides in the cell, what finally leads to cell death1.
The herpes simplex virus thymidine kinase has a less stringent substrate specificity than normal thymidine kinase and thus also metabolises ganciclovir, a guanin analogue. The metabolised ganciclovir is then treated by the cells as guanin but finally inhibits DNA replication by chain termination.
To generate a library of high diversity, we use site directed mutagenesis as well as random mutagenesis with Taq polymerase and manganese.

Dual Selection Procedure

Negative Selection:


In a first step, the created library of variants is grown in presence of the old inducer 3-oxo-C6-HSL and a toxic precursor. Variants whose expression is still induced by the old HSL or have a non-functional repressor (EsaR) express the fusion protein which converts the toxic precursor into a toxin (A), non-responsive repressors stay bound to the DNA and inhibit protein expression (B).
After a certain time, the surviving variants are transfered into culture medium without the toxic precurser and without HSL in order to eliminate the fusion protein.

Positive Selection:


In a second step, these variants now undergo a round of positive selection to select for variants that are responsive to the new HSL 3-OH-C6-HSL.
The bacteria are cultured in medium containing the new HSL and the antibiotic whose resistance is part of the fusionprotein. Inducible variants express the resistance protein and survive (A). Variants that are non-responsive do not express it and can not grow (B).
Afterwards, the variants can be plated and analyzed or undergo further rounds of positive / negative selection to enrich for suitable variants.

Potential Delivery Method of Reporter Strains

The idea to administer genetically modified bacteria in the context of IBD was published by Steidler et al. in 20001. Generally, to use engineered probiotic bacteria as a delivery vector for in vivo produced therapeutic agents has been described multiple times1,1,1,1.
As the stomach and the gastrointestinal tract are rough environments for non-adapted (probiotic) bacteria, we suggest to encapsulate the bacteria in a hydrogel. This protects the bacteria and ensures the recovery of the reporter strain. The method of encapsulation is well known for oral administration in animal models (e.g. Prakash et al.1) and is summarised in several reviews (e.g. Prakash et al. (2008)1 and Tomaro et al. (2012) 1).

References:

  • [1] Kaplan, Gilaad G. "The global burden of IBD: from 2015 to 2025." Nature Reviews Gastroenterology & Hepatology 12.12 (2015): 720-727.
  • [1] Scaldaferri, F., et al. "Gut microbiota molecular spectrum in healthy controls, diverticular disease, IBS and IBD patients: Time for microbial marker of gastrointestinal disorders?." Journal of Crohns & Colitis. Vol. 9. Oxford Univ Press, 2015.
  • [1] Ott, S. J., and S. Schreiber. "Reduced microbial diversity in inflammatory bowel diseases." Gut 55.8 (2006): 1207-1207.
  • [1] Hold, Georgina L., et al. "Role of the gut microbiota in inflammatory bowel disease pathogenesis: what have we learnt in the past 10 years." World J Gastroenterol 20.5 (2014): 1192-1210.
  • [1] Kaur, Nirmal, et al. "Intestinal dysbiosis in inflammatory bowel disease." Gut microbes 2.4 (2011): 211-216.
  • [1] Chichlowski, Maciej, and Laura P. Hale. "Bacterial-mucosal interactions in inflammatory bowel disease—an alliance gone bad." American Journal of Physiology-Gastrointestinal and Liver Physiology 295.6 (2008): G1139-G1149.
  • [1] Laukoetter, Mike G., Porfirio Nava, and Asma Nusrat. "Role of the intestinal barrier in inflammatory bowel disease." World Journal of Gastroenterology 14.3 (2008): 401.
  • [1] Kochar, Nitin I., et al. "Nitric oxide and the gastrointestinal tract." Int. J. Pharmacol 7 (2011): 31-39.
  • [1] Archer, Eric J., Andra B. Robinson, and Gürol M. Süel. "Engineered E. coli that detect and respond to gut inflammation through nitric oxide sensing." ACS synthetic biology 1.10 (2012): 451-457.
  • [1] Green, Jeffrey, Matthew D. Rolfe, and Laura J. Smith. "Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide." Virulence 5.8 (2014): 794-809.
  • [1] UCSF Chimera--a visualization system for exploratory research and analysis. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. J Comput Chem. 2004 Oct;25(13):1605-12.
  • [1] Thompson, Jessica Ann, et al. "Manipulation of the quorum sensing signal AI-2 affects the antibiotic-treated gut microbiota." Cell reports 10.11 (2015): 1861-1871.
  • [1] Landman, Cecilia, et al. "Sa1804 Quorum Sensing Driven by N-Acyl-Homoserine Lactone in Inflammatory Bowel Diseases Associated Dysbiosis." Gastroenterology 144.5 (2013): S-310.
  • [1] Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell 163.3 (2015): 759-771.
  • [1] Shong, Jasmine, et al. "Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity." ACS chemical biology 8.4 (2013): 789-795.
  • [1] Rackham, Oliver, and Jason W. Chin. "A network of orthogonal ribosome· mRNA pairs." Nature chemical biology 1.3 (2005): 159-166.
  • [1] Tominaga, Masahiro, et al. "Rapid and liquid-based selection of genetic switches using nucleoside kinase fused with aminoglycoside phosphotransferase." PloS one 10.3 (2015): e0120243.
  • [1] Hartmann, K. U., and Charles Heidelberger. "Studies on fluorinated pyrimidines XIII. Inhibition of thymidylate synthetase." Journal of Biological Chemistry 236.11 (1961): 3006-3013.

Thanks to the sponsors that supported our project: