Introduction

BNDS-China 2025 experimentally contributed to the part BBa_K811003, INPNC tag, for which we demonstrated its effectiveness in surface display by tagging one of our keratinases and performing super resolution microscopy.

We also characterized fluorescent and chromoprotein libraries from the 2025 iGEM distribution kit and developed a novel feces-mimicking model using curry powder, which is an accessible approach for high school teams unable to work with real cat feces due to ethical considerations.

Building on BNDS-China 2024’s work, we integrated the Tes4 butyrate production module into our project for its potential to alleviate intestinal inflammation associated with IBD. We conducted literature reviews and provided insight into the butyrate production part BBa_K3838613.

INPNC Tag Characterization

INPNC (Penn 2012, BBa_K811003) is a truncated version of the iced nucleation protein (INP), with its N- and C-termini fused to enable localization at the cell surface (Figure 1A-B). The INPNC tag was fused to sfGFP via a 13 amino acid-long flexible GS linker (ELTE 2022, BBa_K4375010). The coding sequence of INPNC-linker-sfGFP was cloned downstream of the IPTG inducible T7 promoter (Figure 1C).

Figure 1. Structural prediction and plasmid design of INPNC surface display system. (A) Predicted structure of iNPNC (Blue) fused to KrMKU3 (Green), generated using ChimeraX. (B) Structural model INPNC-tagged keratinase (purple) anchored in a phospholipid bilyaer, generated by ChimeraX. (C) The INPNC-liner-sfGFP construct cloned into pET28a(+) for the purpose of overexpression. The plasmid was synthesized by Tsingke, and the sequence was confirmed via NGS.

Cells transformed with pET28a(+)-INPNC-linker-sfGFP showed a distinct peripheral ring of fluorescence surrounding a dark intracellular region. (Figure 2C), indicating successful surface localization of sfGFP via the INPNC tag. By contrast, cells harboring the pET28a (+) - sfGFP without the INPNC linker showed uniform intracellular fluorescence, consistent with cytoplasmic localization rather than membrane anchoring. (Figure 2B) These results confirm that the INPNC tag is essential for achieving surface display of sfGFP.

Interestingly, the dotted fluorescent background around the rod-shaped cells likely reflects leakage or release of unanchored fluorescent proteins resulting from overexpression of the INPNC fusion construct. This observation is also consistent with our PI's previous experimental findings reported during the iGEM 2018 project (https://2018.igem.org/Team:BJRS_China/Results)

Figure 2: Super-resolution microscopy of INPNC-mediated surface display. Captured using Opera Phenix Plus High-Content Imaging System, parameters: 63xfluorescent microscopy,excitation:488nm; emission filter: 500-550nm; laser power:100%; exposure time: 100ms (A) Negative control: cells transformed with empty pET28a(+) vector show no detectable fluorescence but only background noise (B) sfGFP control: cells expressing pET28a(+)-sfGFP exhibit uniform intracellular fluorescence; (C) INPNC surface display: cells expressing pET28a(+)-INPNC-linker-sfGFP display a distinct peripheral fluorescence ring surrounding a dark intracellular region, confirming successful surface localization of sfGFP via the INPNC tag. The surrounding fluorescent is the secreted GFP.

BNDS-China 2025 validated the INPNC tag's efficiency of surface display in E. Coli BL21 data.

Color Report Characterization Contributions

We aim to alert pet owners when their cats are in diseased conditions. To achieve this, we designed our probiotic to express fluorescent proteins in response to IBD biomarkers, thereby creating a clear visual cue in the feces.

To characterize and identify the best candidates for color display, this year, we amplified and cloned 8 chromoproteins and 26 fluorescent proteins from 2025 iGEM distribution kit (Figure 1B.) into a constitutive expression vector. (Figure 1A.)

Figure 3. Plasmid design of the color signal module and the proteins involved Plasmid design of color signal module. Created by biorender.com.

After screening under UV light for fluorescent proteins and under normal light for those chromoproteins, we selected 9 proteins for further testing: SYFP2, Tannen RFP, amilRP, spisPink, aeBlue, gfasPurple, amilCP, eechGFP3, and afraGFP.

To meet the need for visualizing the colored signals in cats' feces, the second round of screening was performed by mixing fluorescent proteins with curry powder.

Figure 4. 2.0 grams of curry powder, 100 uL of soy sauce, and 200uL of water to mimic the appearance of cat feces.

Our results indicated that amilRP and gfasPurple exhibited the most significant color change, and afraGFP produced the most visible signal in the curry mixture.

Figure 5. Characterization of fluorescent and chromoproteins (A) Colony appearance under visible and UV light after cloning into constitutive expression vector; (B) color visualization of selected proteins mixed with the curry feces mimic under normal light; (C) Fluorescent visualization under UV light.

Here, BNDS-China contributed to the characterization of existing fluorescent protein and chromoprotein library, and we demonstrated the potential use of afraGFP in visualization in future iGEM project. We also provide an accessible approach for mimicking feces using curry powder, which is particularly helpful for high school teams that cannot work with real cat feces due to ethical concerns.

Tes4 Butyrate Production Contribution

We conducted a literature review and contributed to the documentation of Tes4-related parts (BBa_K3838613). In 2024, BNDS-China demonstrated that Tes4 enables the production of butyrate and successfully quantified butyric acid yield in an E. coli chassis.

Building on this foundation, our project shifts the application context from human health to animal health. From our review, we found that butyric acid is also implicated as a biomarker associated with the presence of IBD, highlighting its potential relevance in our animal-focused study.

Figure 6. Landscape of our project design, with highlighting in butyric acid anti-inflammatory module. The red box highlights the incorporation of butyrate as a key output of our therapeutic platform.

Recent studies in cats with chronic enteropathy, including IBD, have shown significant alterations in the gut microbiome, with decreased bacterial diversity and reduced abundance of butyrate-producing taxa such as Ruminococcaceae and Bifidobacteriaceae, while opportunistic families like Enterobacteriaceae and Streptococcaceae were enriched. Because butyrate is a major energy source for colonocytes and exerts anti-inflammatory effects that help maintain intestinal barrier integrity, its depletion has been proposed as a mechanistic link between dysbiosis and feline IBD. [1]

Butyric acid is not only implicated as a biomarker of intestinal inflammation but has also been shown to exert protective effects on feline gut health. A recent multi-omics study in adult ragdoll cats demonstrated that dietary supplementation with 0.1% sodium butyrate significantly reduced pro-inflammatory cytokines IL-1beta and tumor necrosis factor-α (TNF-α), enhanced antioxidant capacity, and improved intestinal barrier integrity by decreasing fecal calprotectin levels. [2]

Together, this evidence highlights that supplementing butyrate production may not only serve as a biomarker of disease but also provide a therapeutic benefit, making Tes4-based butyrate synthesis highly relevant in the context of feline gut health.

References

↑ Marsilio, S., Pilla, R., Sarawichitr, B., Chow, B., Hill, S. L., Ackermann, M. R., Estep, J. S., Lidbury, J. A., Steiner, J. M. & Suchodolski, J. S. (2019). Characterization of the fecal microbiome in cats with inflammatory bowel disease or alimentary small cell lymphoma. Scientific Reports, 9(1), 19208. https://doi.org/10.1038/s41598-019-55691-w

↑ Zhang, A., Li, D., Yu, T., Zhang, M., Cui, Y., Wang, H., Dong, T. & Wu, Y. (2025). Multi-Omics Approach to Evaluate Effects of Dietary Sodium Butyrate on Antioxidant Capacity, Immune Function and Intestinal Microbiota Composition in Adult Ragdoll Cats. Metabolites, 15(2), 120. https://doi.org/10.3390/metabo15020120

↑ Penn, J. (2012). INPNC tag for surface display. iGEM Registry. https://parts.igem.org/Part:BBa_K811003

↑ ELTE (2022). GS linker for protein fusion. iGEM Registry. https://parts.igem.org/Part:BBa_K4375010