Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Microbial Biosensor for Sensing and Treatment of Intestinal Inflammation.

doi: 10.1002/advs.202504364

Figure Lengend Snippet: Figure 4. Detection of calprotectin as an intestinal inflammation biomarker by BSIM. (A) Experimental design for testing BSIM in DSS-induced gut inflammation mouse model (five female and five male mice in each group). (B) Concentration of calprotectin in mouse colon contents. Colon contents at the end of the experiment were isolated and tested for calprotectin levels by ELISA. Data are median ± interquartile range, n = 10; Individual dots represent individual mice. Statistical analysis was performed using Kruskal–Wallis test; ***p < 0.001, ****p < 0.0001. (C) Fold activation of BSIM in mouse colon contents. Data are median ± interquartile range; for in vitro control n = 3 biological independent repeats, for in vivo experiments n = 10, individual dots represent individual mice. Statistical analysis was performed using Kruskal–Wallis test; **p < 0.01, ****p < 0.0001. (D) Correlation analysis of biosensor BSIM activation and colon calprotectin concentration. n = 40; individual dots represent individual mice. Correction analysis was performed by computing Pearson correlation coefficients with 95% confidence interval and two-tailed p-value analysis. (95% CI = 0.616–0.877, r = 0.778, p < 0.0001). (E) Experimental design for testing BSIM in C. difficile induced gut inflammation mouse model (for in vivo control group that treated with antibiotics but without C. difficile infection n = 10 with 5 female and 5 male mice, for C. difficile infection group n = 20 with ten female and ten male mice). (F) Concentration of calprotectin in the mice colon contents. Colon contents at the end of the experiment were isolated and tested for calprotectin levels by ELISA. Data are median ± interquartile range; Individual dots represent individual mice, for in vivo control group n = 10, for C. difficile infection group n = 15 (five mice died or difficult to get samples). Statistical analysis was performed using Mann–Whitney test; ****p < 0.0001. (G) Fold activation of BSIM in mouse colon contents. Data are median ± interquartile range; Individual dots represent individual mice, for in vivo control group n = 10, for C. difficile infection group n = 15 (five mice died or difficult to get samples). Statistical analysis was performed using Mann–Whitney test; ****p < 0.0001. (H) Correlation of biosensor BSIM activation and colon calprotectin concentration. n = 25; Individual dots represent individual mice. Correction analysis was performed by computing Pearson correlation coefficients with 95% confidence interval and two-tailed p value analysis. (95% CI = 0.798–0.959, r = 0.907, p < 0.0001).

Article Snippet: Supernatants of therapeutic biosensor cultures with TPEN induced were analyzed by IL10 ELISA kit (EAGLE Biosciences, Nashua, NH).

Techniques: Biomarker Discovery, Concentration Assay, Isolation, Enzyme-linked Immunosorbent Assay, Activation Assay, In Vitro, Control, In Vivo, Two Tailed Test, Infection, MANN-WHITNEY

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Microbial Biosensor for Sensing and Treatment of Intestinal Inflammation.

doi: 10.1002/advs.202504364

Figure Lengend Snippet: Figure 6. Engineering therapeutic biosensors to secret functional secIL10 (YebF-IL10). (A) Diagram of therapeutic sensor plasmid pBSIDZ2. The yebF gene from EcN was fused into the N-terminal of IL10 (referred to as secIL10) and placed under the control of Pykg (secIL10 replaced sfgfp gene in pBSI2), resulting in therapeutic strain PRB5002. The yebF expression control strain PRB5001 was used as a control. (B) YebF facilitates secIL10 secretion in EcN. Biosensor PRB5002 (EcN/PBSIDZ2) was cultured to OD600 of 0.5–0.6 and then induced with 20 μM TPEN for 4 h in LB media. Following, the supernatants of cultures were collected and detected by IL10 ELISA kit. PRB5001 (EcN/PBSIDZ1) which expresses YebF only was used as a control. Data are mean ± SEM, n = 4 biological independent repeats. Statistical analysis was performed using an unpaired t-test; ****p < 0.0001. (C) secIL10 exhibits functional IL10 activity in HEK-Blue IL10 reporter cell line. Quantified secIL10 from PRB5002 supernatant was added to HEK-Blue IL10 reporter cells line to activate reporter expression. Recombinant human IL10 was used as a positive control. Supernatants from PRB5001 (EcN/PBSIDZ1) with only YebF were used as a negative control. Data are mean ± SEM, n = 4 biological independent repeats; Statistical analysis was performed using ANOVA Tukey test; *p < 0.05, ****p < 0.0001. (D) Diagram of secIL10 stable expression sensor BSIT. The asd gene with its own promoter from EcN was cloned into pBSIDZ2 (referred to as pBSIT), following was transformed into EcNΔasd, resulting in stable secIL10 expression therapeutic strain BSIT (EcNΔasd/pBSIT). Control strain BSIC which only expresses YebF was used as a control. (E) BSIT exhibits plasmid stability in the absence of antibiotic selection. Therapeutic biosensors PRB5002 (EcN/pBSIDZ2) and BSIT (EcNΔasd/pBSIT) were cultured in LB media or LB+10 μM TPEN without antibiotic selection, respectively. The biosensors were passaged five times, following 100 μL of ten-fold serial dilution cultures were plated on LB agar plates, then a single colony was replica-cultured in LB and LB with 15 μg mL−1 chloramphenicol agar plates to calculate plasmid stability. Data are mean ± SEM, n = 3 biological independent repeats. Statistical analysis was performed using an unpaired t-test; ****p < 0.0001. (F) Diagram of therapeutic sensor plasmid (pBSIMT) with memory circuit. secIL10 replaced sfgfp gene in pBSIM2 with Pasd-asd (referred to as pBSIMT), following pBSIM1 and pBSIMT were transformed into EcNΔasd, resulting in memory circuit therapeutic strain BSIMT. Control strain BSIC which only expresses YebF was used as a control. (G) Secretion of secIL10 in biosensor BSIT and BSIMT. Biosensors were cultured to OD600 of 0.5–0.6 and then induced with 20 μM TPEN for 4 h in LB media. The supernatants of cultures were collected and detected by IL10 ELISA. The expression of secIL10 in BSI1 and BSIM remains similar, indicating no impact on IL10 expression with the addition of asd to the plasmid. Data are mean ± SEM, n = 3 biological independent repeats. Statistical analysis was performed using one-way ANOVA with the Tukey test; ****p < 0.0001.

Article Snippet: Supernatants of therapeutic biosensor cultures with TPEN induced were analyzed by IL10 ELISA kit (EAGLE Biosciences, Nashua, NH).

Techniques: Functional Assay, Plasmid Preparation, Control, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay, Recombinant, Positive Control, Negative Control, Clone Assay, Transformation Assay, Selection, Serial Dilution

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Microbial Biosensor for Sensing and Treatment of Intestinal Inflammation.

doi: 10.1002/advs.202504364

Figure Lengend Snippet: Figure 7. Biosensor coupling with secIL10 ameliorates intestinal inflammation. (A) Experimental design of therapeutic biosensors applied to 3% DSS- induced gut inflammation for the treatment of intestinal inflammation. Each group has ten male mice. (B) Percentage of mice with bloody feces. Mice producing bloody feces were monitored and recorded every day for 11 days. Groups tested include DSS + BSIT, DSS + BSIMT, DSS + BSIC (control expressing only YebF), DSS + BSIMC (control expressing only YebF), and DSS + PBS (colitis control). Groups were compared to animals that received only gavage of PBS without DSS treatment. (C) Colon length. Data are presented in a box & whiskers plot with min to max, showing all points, n = 10, and individual dots represent individual mice. Statistical analysis was performed using Kruskal–Wallis test; *p < 0.05, **p < 0.01, ***p < 0.001. (D) Histological score of colon tissue. Mouse distal colon tissue was collected, fixed, sliced, stained (H&E staining), and scored. Data are median ± interquartile range, n = 10; Individual dots represent individual mice. Statistical analysis was performed using Kruskal–Wallis test; *p < 0.05. (E) Representative hematoxylin and eosin (H&E) stained images of mouse distal colon sections from each experimental group. Black arrows: inflammatory cell infiltrates in the mucosa and submucosa. Double-headed black arrow: lamina propria expansion. Red arrows: loose of connective tissue. Green arrows: disappearance of glands and goblet cells. (F) Concentration of calprotectin in mice feces. Mice fecal samples were collected on day 11 and tested for calprotectin concentration by ELISA. Data are median ± interquartile range, n = 10; Individual dots represent individual mice. Statistical analysis was performed using Kruskal–Wallis test; *p < 0.05. (G) Concentration of MCP-1 (Monocyte Chemoattractant Protein-1) in mice serum. Mice serums were collected on day 11 and tested for MCP-1 concentration by ELISA. Data are median ± interquartile range, n = 10, Individual dots represent individual mice. Statistical analysis was performed using Kruskal–Wallis test; *p < 0.05.

Article Snippet: Supernatants of therapeutic biosensor cultures with TPEN induced were analyzed by IL10 ELISA kit (EAGLE Biosciences, Nashua, NH).

Techniques: Control, Expressing, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay