Project Description

Background Information

Domestic cats are among the most beloved companion animals in households worldwide with a population exceeding 220 million (Jergens, 2012). Despite their popularity, feline health is often overlooked, largely due to insufficient studies in veterinary science and subtle disease symptoms at early stages. Unlike humans, cats cannot articulate their discomfort, and many signs of illness may go unnoticed by their owners. This delay in recognition often leads to missed opportunities for timely intervention, placing cats at risk of developing severe or chronic conditions.

One of the severe diseases is Inflammatory Bowel Disease (IBD), which has the potential to cause vomiting, diarrhea, and weight loss in addition to inflammation in multiple organs (Jergens, 2012). IBD is often correlated with hairball accumulation in the feline gut, which forms a positive feedback loop and collectively worsens the intestinal condition. The accumulation of hairballs impacts feline digestive and absorptive abilities and may be further amplified by IBD. (Cornell Feline Health Center, 2025)

Problems Faced

Current treatments of cat IBD include dietary modifications, pharmacologic therapies, and immunosuppressive drugs (Jergens, 2002). Due to the variety of potential causes and the complex nature of IBD in cats, however, these approaches often suffer from limited precision and lack long-term efficacy (Jergens, 2012). Dietary interventions require extended trial periods and may not yield consistent results across individual cats. Traditional medications require systemic drug intake, which can cause side effects and disrupt gut microbiota. Immunosuppressive drugs carry additional risks, such as impaired blood cell production and increased susceptibility to infections, and must be administered with caution (Willard, 1999).

Moreover, the above therapy strategies fail to address hairball accumulation, which is a critical compounding factor in feline IBD. Hairballs worsen the IBD conditions, and IBD further slows the degradation of hairballs, forming a positive feedback loop (Cornell Feline Health Center, 2025). One existing treatment for hairballs is hairball pastes, acting as lubricants to facilitate the removal of hairballs; however, this could be problematic. Many hairball pastes contain mineral oils (such as paraffin oil) or vegetable oils (such as soybean oil) for lubrication, which might cause fat-soluble vitamin deficiencies, dehydration, and fecal incontinence in addition to impacting gut microbiota and digestive enzyme activities in mammals (Qiao et al., 2022). Moreover, some hairball pastes also contain sweeteners to attract cats, which increases the risk of diabetes. Finally, the usage of hairball pastes may obscure symptoms of other cat diseases, such as gastrointestinal dysfunction and chronic constipation, and may delay the process of detection and treatment of other diseases.

The problems identified above emphasize the need for a novel treatment method to combat IBD in cats.

Our Solution

Our project aims to engineer an in vivo, long-term effective, and multi-potent probiotic platform, which functions to detect, treat, and record IBD in the feline gut.

Figure 1. Overview of our project design. Created by biorender.com.

In our platform, we apply the intestinal food input as an overall on-off switch of the whole system, ensuring that all downstream pathways are activated only after food uptake. Since food provides the essential energy and organic materials required for metabolic activity, this mechanism prevents our platform from producing therapeutic enzymes in the absence of sufficient resources, thereby reducing the metabolic burden on the probiotics. To achieve this, we chose to sense bile, which is secreted during digestion and thus serves as a signal of food uptake. Specifically, the major bile acid in the feline gut, taurocholic acid (TCA), was selected as our indicator of food intake (Washizu et al., 1990). For the detection of IBD symptoms in vivo, we choose calprotectin, the gold standard biomarker for clinical diagnosis of IBD (Jukic et al., 2021). Since food input and IBD biomarker should both be detected for the activation of downstream effectors to treat IBD, we adopted an AND gate to integrate these two signals. The outputs of our platform will then be activated when the AND gate is turned on. (Figure 1)

Our output module consists of three parts: butyrate production, hairball degradation, and color signal. Butyrate is an anti-inflammatory short chain fatty acid (SCFA) mostly produced by anaerobic bacterial fermentation (Singh et al., 2023). It is found in low concentration in IBD conditions and has been shown to maintain intestinal homeostasis due to its anti-inflammatory effects on the intestinal epithelium (Marsilio et al., 2019). In the presence of food and calprotectin, our probiotics will produce butyrate to treat IBD. Moreover, to further tackle the hairball accumulation problem due to IBD, our probiotics will also express keratinase to degrade keratin, the major protein in cat hairballs. Considering that keratin is a large polymer that might be unable to transport into the cytoplasm, we further adopt a surface display system to secrete keratinase onto the E. coli cell surface for a higher degradation rate. Lastly, the platform will bring bright colors in cat feces through fluorescent proteins and chromoproteins in cases of IBD conditions, which, when integrated with an automated cat litter box, can notify the cat owner when diseases occur. (Figure 1)

Although our platform can achieve long-term monitoring and treatment of IBD, it is also crucial to track the history of IBD development for more detailed and individualized treatments for the cat in the future. Our platform does so by recording the activities of promoters-of-interest and permanently storing the information into the bacterial genome. In cases where the disease becomes too severe or too complex to be handled by our probiotic platform, the veterinary surgeon could choose the most effective treatment based on the past activities recorded by gut probiotics. (Figure 1)

Compared to existing treatment options, our probiotic platform has several improvements. First, our treatment is based on E. coli Nissle 1917 (EcN) as the platform, which is a well-established commercial probiotic (Snell et al., 2022). This creates fewer side effects than the traditional methods like external drug intake. Second, our probiotic targets both IBD biomarker and hairball accumulation, so that it can effectively inhibit their positive feedback loop and lower the likelihood of future recurrence. Also, our probiotic platform is able to achieve long-term monitoring and treatment of the disease, instead of single-dose drug administrations. Lastly, the visual display and data storage modules can report the progression of disease in a timely manner through a strong visual signal, as well as to record the trajectory of disease progression, allowing a timely and individualized intervention.

Information Input Module

Food Input Detection

Taurocholic acid (TCA) is the most abundant component in feline gallbladder bile, accounting for over 50% of the total bile acid content (Washizu et al., 1990). We therefore consider it as a strong indicator of food intake activity. During digestion, TCA is secreted into the duodenum and remains at high concentrations until it is largely absorbed in the ileum (Summers and Quimby, 2024). This enables the interaction of TCA with bacterial biosensors in the small intestine of cats, ensuring the effective functioning of our design.

We designed and tested two systems to detect TCA. The first system is based on its interaction with CmeR, a TetR-family transcriptional repressor originally identified in the Gram-negative bacterium Campylobacter jejuni (Lin et al., 2005). Multiple bile acids have been found to interact with CmeR and disrupt the binding between CmeR and its operator sequence cmeO in C. jejuni, and the binding with TCA is the strongest among them (Lei et al., 2011). In the structural perspective, CmeR forms a homodimer structure and binds to its operator DNA through the helix-turn-helix motif at its DNA-binding domain (Figure 2A). The binding of TCA triggers a conformational change in CmeR homodimer, weakening its DNA-binding affinity (Figure 2B, C). CmeR has been heterologously expressed and well-characterized as a transcriptional regulator in E. coli; it has shown inducibility by salicylate (8-fold) and other 21 molecules, but the response to TCA has never been tested in E. coli (Nasr et al., 2022).

Figure 2. The CmeR structure predicted by Alphafold 3. A, The homodimer structure (chain A: cyan; chain B: green) of CmeR binds to the cmeO DNA region. B, The ligand-bound structure of CmeR homodimer (chain A: cyan; chain B: green) when bound with TCA (pointed by red arrows). C, Structural alignment of CmeR DNA-bound structure (green) and ligand-bound structure (cyan); a structural shift was observed at the helix-turn-helix DNA binding domain (red circled).

However, the first system only senses cytoplasmic TCA and is limited by TCA's poor membrane permeability (Elkins & Savage, 2003). We therefore designed and tested the second system based on the transmembrane sensor TcpP, which is able to detect and respond to TCA outside the membrane. TcpP is responsible for bile salt sensing in Vibrio cholerae; in the presence of ligands, TcpP dimerizes and acts as a transcriptional factor, therefore activating downstream gene expression (Xue et al., 2016). Its subcellular localization (on the membrane with its ligand-binding domain oriented towards the extracellular side) makes it compatible with our need for the TCA sensor and addresses the problem of low membrane permeability of TCA.

The above TcpP-based TCA sensor has shown high compatibility to E. coli after being integrated into a previously well-characterized, widely-applicable synthetic receptor platform called EMeRALD (short for Engineered Modularized Receptors Activated via Ligand-induced Dimerization). This TcpPH-EMeRALD system demonstrated its ability to sense TCA with high sensitivity (with the EC50 of 28.344 μM) and decent dynamic range (84.92-fold) in E. coli, making it suitable for our platform design. (Chang et al, 2021)

IBD Biomarker Detection

We designated calprotectin as the IBD biomarker in our platform. Calprotectin is the clinical gold standard IBD biomarker due to its non-invasiveness, specificity, and sensitivity targeting gut inflammation. Calprotectin levels rise significantly during inflammatory conditions, which shows its ability to reflect the disease condition. (Jukic et al., 2021)

Since calprotectin can bind with zinc and therefore reduce the free zinc ion concentration inside the probiotic, we use a zinc-responsive promoter found in EcN, ykgMO (BBa_K5180004), to sense abnormal elevations in calprotectin concentration. This promoter is under the control of repressor Zur in the EcN genome, whose activation requires binding with free zinc ions. (Figure 3) Past research has characterized the promoter's robustness for calprotectin detection over multiple media conditions (a 21.2-fold change in M9 media and a 16.9-fold change in LB media). Moreover, this ykgMO-based calprotectin system could successfully sense intestinal inflammation in mice, suggesting the promoter's ability to function well in mammal gut environments. (Xia et al., 2023; Zhu et al., 2025)

Figure 3. The zinc-bound structures of Zur and calprotectin predicted by Alphafold 3. A, Zur. B, calprotectin.

Information Processing Module

AND gate is the core of our system’s logic design that integrates two critical physiological inputs: TCA signaling food intake, and calprotectin indicating IBD-related inflammation. This dual-input architecture ensures that therapeutic responses are only activated under precise conditions, when the cat has food serving as the energy source and IBD symptoms requiring treatment. This logic gate avoids unnecessary enzyme expression in healthy or starved states, reducing the gene expression burden on our probiotics. (Figure 4)

Figure 4. Overall design of the AND gate. Created by biorender.com.

To apply this logic gate to our project, we explored two distinct architectures acting at different regulatory layers. The first system was inspired by a previous design (Stanton et al., 2014), in which we implemented the AND logic gate at the transcriptional level through the plasmid pAND. This design relies on the modular combination of multiple prokaryotic transcriptional repressor-promoter pairs, enabling the construction of composite logic gates in E. coli. For pAND, orthogonal transcriptional NOT gates (built from repressors PhIF and QacR) are used to invert the two input promoters, and the inverted outputs are combined into a NOR gate (built from the repressor BetI) that controls the downstream reporter. This combined logic implements the AND logic gate. Thus, only when two input signals are both present, will the output be activated. (Figure 5)

Figure 5. Schematic and the truth table of pAND. Created by biorender.com.

Our second AND gate system was the split T7 RNA polymerase (RNAP) system originally developed by Schaerli et al. (2014). In this design, the T7 RNAP is divided into N- and C-terminal fragments, each fused to an intein domain. When both fragments are expressed, the inteins mediate protein splicing, reconstituting a functional T7 RNAP. Because enzymatic activity arises only upon the co-expression and assembly of both protein fragments, this is considered a post-translational AND gate strategy. The reconstituted polymerase then drives transcription from a T7 promoter, thereby enabling a protein-interaction-based control of transcriptional activity. Compared with pAND, the split T7 RNAP system might offer an expanded dynamic range and lower leakage. Most importantly, the system was implemented in probiotic E. coli strain Nissle 1917, which lacks endogenous T7 RNAP. This ensures orthogonal transcription, with output occurring exclusively through the split T7 RNAP system, further minimizing basal expression and making it a promising AND gate candidate.

Functional Output Module

Anti-inflammatory Production

The first part of our output module is to synthesize an anti-inflammatory molecule that directly combats IBD. Butyrate was selected for its exceptional anti-inflammatory properties in feline models. Previous studies have demonstrated that even low-level dietary supplementation with sodium butyrate (0.1%) could reduce IBD-related biomarkers such as pro-inflammatory cytokines (TNF-α, IL-1β), as well as modulating gut microbiota by increasing probiotics like Lachnospiraceae and Roseburia, suggesting its high potential as a therapeutic for feline IBD. (Zhang et al., 2025)

For butyrate production, we utilize the native FASII pathway and simplify this biosynthesis process into a single-enzyme approach, which significantly reduces the metabolic burden of our probiotic platform. Tes4 (BBa_K3838613), an acyl-ACP thioesterase from Bacteroides fragilis, catalyzes the production of butyric acid from butyryl-ACP (an intermediate in the FASII pathway) to enable butyrate production. (Figure 6) Prior studies have validated the successful expression and enzymatic activity of Tes4 in various E. coli strains (Jing et al., 2011; Kallio et al., 2014), supporting its compatibility and anticipated efficacy in our EcN-based probiotic system.

Figure 6. Butyrate production based on the FASII pathway and Tes4 enzyme.

Keratin Degradation

Keratin is the major protein component of hairballs. It exists in two major fibrous forms: α-keratin, with an α-helical structure commonly found in mammalian hair and wool, and β-keratin, composed of β-pleated sheets and present in reptiles and birds. Cat fur, primarily composed of α-keratin, exhibits high tensile strength and chemical stability due to extensive disulfide cross-linking between cysteine residues. Keratinases degrade keratin through diverse mechanisms and substrate specificities that depend on the enzyme structure. (Nnolim et al., 2020)

For our proposed application in the feline gastrointestinal tract, the keratinase in our final implementation should exhibit high keratinolytic activity specific to α-keratin, operate within pH and temperature ranges compatible with the feline gut environment, and avoid eliciting immune responses or damaging the intestinal epithelium (Sato et al., 2019). To achieve those, we selected four keratinases based on their stability, keratinolytic activity, substrate specificity, and safety profiles.

The first keratinase is KrMKU3, originally isolated from Bacillus megaterium (Radha & Gunasekaran, 2007). It was successfully expressed in E. coli by the previous iGEM team SZ-SHD 2021 (BBa_K3895005), and its activity was confirmed by in vitro human hair degradation assays. The second keratinase was derived from Bacillus sp. LCB12 by Tian et al. (2019), and is named by us as KrLCB12. It has demonstrated degradation activity on α-keratin-rich wool (which is similar to cat fur), although it has not yet been expressed in E. coli. (Tian et al., 2019) The third keratinase, KrOcL9, was identified from Bacillus sp. OcL9. In addition to successfully degrading α-keratin, this enzyme showed no detectable degradation of collagen types I and II, therefore less likely to damage the feline gut interior. (Li et al., 2022) Our fourth and last keratinase is KrUS575, sourced from Brevibacillus brevis US575. It was chosen because it exhibits optimal activity at pH 8 and 40°C, conditions that closely match the environment of the feline gut. (Jaouadi et al., 2013)

Considering the larger size of the hairball compared to the probiotic cells, the degradation process must be directly in the intestinal lumen rather than inside the bacterial cytoplasm. Therefore, we employ a surface display system using INPNC from the previous iGEM team Penn 2012 (BBa_K811003), a truncated version of the ice nucleation protein (INP) derived from Pseudomonas syringae. INPNC retains the N- and C-terminal membrane-anchoring domains of INP but lacks the central repeated domain and has been widely validated for outer membrane protein display in E. coli. (Figure 7) By fusing the keratinase to INPNC, we can achieve cell surface localization of the enzyme, allowing direct interaction with extracellular keratin substrates in the gut environment.

Figure 7. INPNC prediction and structural modeling. A, AlphaFold3-predicted INPNC structure. B, AlphaFold3-predicted INPNC structure anchored within the phospholipid bilayer.

Color Visual Display

Upon sensing biomarkers associated with intestinal inflammation in feline feces, our designed probiotic platform will start expressing fluorescent proteins, therefore bringing bright colors in cats' feces as a visual signal of disease conditions. This fluorescence serves as a direct, non-invasive visual signal that alerts cat owners to potential disease conditions, enabling earlier diagnosis and timely intervention. Unlike current pharmacologic therapies, which lack the capability to provide rapid, real-time feedback on the cat's intestinal health, our color visual output module offers an immediate and intuitive diagnostic cue.

This visual display module uses fluorescent proteins and chromoproteins for presentation. We expressed 9 chromoproteins and 25 fluorescent proteins cloned from the iGEM distribution kit (Figure 8). After testing all of them under natural and UV lights, we then picked 9 proteins with the highest visibility and tested them in fake cat feces.

Figure 8. Proteins included in our color visual display module.

Data Storage Module

Feline gut health remains poorly characterized due to insufficient in situ, time-based data. To address this gap, we aim to construct a transcriptional recording system in engineered gut-resident probiotics, capable of capturing gene expression events associated with IBD. Such a system would enable longitudinal profiling of microbial responses and support further research studies and the development of individualized veterinary interventions.

Among various recording strategies, the Retro-Cascorder system offers the advantage of selectively logging via signal-specific barcode incorporation rather than the entire transcriptomic activity. The Retro-Cascorder works by 1) converting transcribed retron non-coding RNA into a DNA barcode using a retron reverse transcriptase, and 2) storing that event in a unidirectionally expanding CRISPR array via acquisition by CRISPR-Cas integrases Cas1-Cas2. (Lear et al., 2023) This CRISPR array-based recording of gene expression can be later retrieved by high-throughput sequencing. Hence, this system can chronologically record IBD-related transcriptional events of promoters-of-interest, which enables veterinarians to provide precise and targeted medication dependent on the recorded disease progression.

Aiming for better genome insertion efficiency of our recorder system, we further constructed and tested three additional Cas1 variants identified by Yosef et al. (2023): Cas1(E296G), Cas1(V76L), and Cas1(V76L E269G). We will incorporate the Cas1 variant with the highest insertion into our platform.

However, this design of the Retro-Cascorder system is only functional for E. coli BL21(AI) strain and not for EcN, on which our probiotic platform will be based. One of the reasons is the absence of an endogenous CRISPR array in the EcN genome. To eventually incorporate this data storage module into our platform, we addressed this problem by integrating the native CRISPR array from BL21(AI) genome into the EcN genome. We utilized the CRISPR Cas9 and Lambda Red recombination system proposed by Li et al. (2021) to integrate the CRISPR array, and our result showed successful integration.

Proposed Implementation

To provide an end-to-end health monitoring service in coordination with our intestinal probiotic platform, we designed an intelligent litter box that integrates automatic cleaning with fluorescence-based disease detection. The system detects IBD in cats by identifying specific fluorescent proteins in their feces and transmits results to a digital interface for owner notification.

The litter box combines a UV-assisted detection module with an automated cleaning mechanism, enabling both precise health monitoring and hygiene maintenance. Together, these functions create a closed-loop system for pet health management, linking gut-level biosensing with daily household monitoring (more details are described in our Hardware section).

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