Part:BBa_K5378012

TFF3

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

To create therapeutic curli fibers, we plan to attach TFF3 (Trefoil Factor 3) to the curli fibers naturally secreted by E. coli to reduce TFF3's adhesion to the intestinal mucus layer. This will maximize TFF3's ability to repair the intestinal barrier and reduce inflammation. Effective prevention and treatment of abdominal infections in liver disease patients, addressing dysbiosis, and specifically repairing common intestinal issues will enhance the safety and efficacy of the engineered bacterium. For this purpose, we referred to a study published in Nature Communications And the PATCH system was used for plasmid design. We first linked the gene fragments responsible for expressing curli fibers to the PBbB8k plasmid, then introduced a 6xHis-tagged linker to connect curli fibers with TFF3, and finally incorporated the TFF3 gene fragment. This configuration allows EcN to secrete and self-assemble curli fibers, linkers, and TFF3 upon reaching the intestine, forming an active domain layer on the intestinal surface. This promotes epithelial cell migration, reduces inflammatory factor levels, supports intestinal barrier repair, and alleviates hepatic encephalopathy complications.

Usage and Biology

For the safety module,we referred to a study published in Nature Communications And the PATCH system was used for plasmid design. We first linked the gene fragments responsible for expressing curli fibers to the PBbB8k plasmid, then introduced a 6xHis-tagged linker to connect curli fibers with TFF3, and finally incorporated the TFF3 gene fragment. This configuration allows EcN to secrete and self-assemble curli fibers, linkers, and TFF3 upon reaching the intestine, forming an active domain layer on the intestinal surface. This promotes epithelial cell migration, reduces inflammatory factor levels, supports intestinal barrier repair, and alleviates hepatic encephalopathy complications.

Figure 1.our safety module design section

Functional Verification

From the figure below, the size of each band of agarose gel electrophoresis is basically the same as the size of the target gene, indicating that the plasmid has been successfully transformed into Ecn.

Figure 2.Transfer to bacterial pcr results with TFF3 plasmids

In order to confirm that curli fibers decorated with TFFs could be produced by EcN, as they can in laboratory strains of E. coli, we transformed EcN with the panel of synthetic curli plasmid constructs (Fig.3-a), in addition to a vector in place of the curli genes as a negative control. The transformed cells were cultured at 37 °C and induced with L-(+)-arabinose.

The secretion of TFF3 can be detected by Mouse trefoil factor 3(TFF3) enzyme-linked immunosorbent Assay kit. Results show that the engineered EcN was strongly induced by L-(+)-arabinose with twice as much TFF3 is produced comparing to no induction (Fig3-b).

The secretion of TFF3-fused curli was proved successful (Fig.3-c), however, In some cases, basal expression of the csgA genes was observed without induction.

A quantitative Congo Red-binding (CR) assay, normally used for curli fiber detection, indicated that CsgA-TFF3 fusions could be expressed and assembled into curli fibers under these conditions, while EcN control vector showed no CR binding(Fig3-d).

Figure 3.Functionality verification of the PEA-sensing NH3-metabolizing system.(a)Schematic representation of the process of sensing and metabolic module. EcN was co-transformed with plasmid Pcon-FeaR-Pcon-TynA and plasmid PTynA-GS via electroporation. (b)NH3 concentration after coculturing different concentration of PEA and NH4Cl with engineered EcN for 12 hours. Data shows mean±SD, n=3 independent experiments.(c)NH3 concentration after coculturing 100ng/ml PEA and 50μM NH4Cl engineered EcN for 0, 4, 8,12 and 24 hours. EcN-FeaR-TynA was transformed with only plasmid Pcon-FeaR-Pcon-TynA as the control group. Data shows mean±SD, n=3 independent experiments.

Reference

[1] Lachar, J., & Bajaj, J. S. (2016). Changes in the Microbiome in Cirrhosis and Relationship to Complications: Hepatic Encephalopathy, Spontaneous Bacterial Peritonitis, and Sepsis. Seminars in liver disease, 36(4), 327–330. https://doi.org/10.1055/s-0036-1593881

[2] Girleanu, I., Trifan, A., Huiban, L., Muzica, C., Nemteanu, R., Teodorescu, A., Singeap, A. M., Cojocariu, C., Chiriac, S., Petrea, O., Zenovia, S., Nastasa, R., Cuciureanu, T., & Stanciu, C. (2021). The Risk of Clostridioides difficile Infection in Cirrhotic Patients Receiving Norfloxacin for Secondary Prophylaxis of Spontaneous Bacterial Peritonitis-A Real Life Cohort. Medicina (Kaunas, Lithuania), 57(9), 964. https://doi.org/10.3390/medicina57090964

[3] Duraj-Thatte, A. M., Praveschotinunt, P., Nash, T. R., Ward, F. R., Nguyen, P. Q., & Joshi, N. S. (2018). Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins. Scientific reports, 8(1), 3475. https://doi.org/10.1038/s41598-018-21834-8