Origin v8

The kinetic parameters of each GOx variant were determined using triplicate ABTS assays in microtiter plates with different glucose concentrations (1.2–266 mM) at pH 5.5 and 7.4. To determine the K_m and k_cat values, the slope of each measurement was calculated over the linear region and fitted to a Michaelis-Menten hyperbola using Origin v8 (OriginLab, Northampton, MA, USA). Lineweaver-Burk, Eadie-Hofstee and Hanes-Woolf plots were also constructed, and the outliers were identified and removed. Protein concentration was determined by measuring the absorbance at 280 nm (the absorption of 1.5 mg/mL GOx is considered equivalent to 1 AU based on the sequence, as calculated using ProtParam).

All DLS, ζ-potential, and plating (cell enumeration) experiments were replicated three times in identical fashion over differing days (N = 3). Additionally, SEM imaging was completed for three identically prepared independent sets of samples over three differing dates. Statistical analysis of data was completed using ORIGIN® v.8 software (OriginLab Corp., Northampton, MA, USA). All microbiological data were log₁₀-transformed prior to statistical analysis. One-way analysis of variance (ANOVA) with Tukey’s Honestly Significant Differences (HSD) post hoc test was used to determine significant differences in data between the treatments at a significance level of P < 0.05.

Purified rcasIL-10R1 was immobilized on a CM5 chip (General Electrics, Uppsala, Sweden) in a Biacore T100 analyzer (General Electrics) in accordance with the manufacturer's recommendations. Briefly, 800 μL of rcasIL-10R1 (0.5 μg/mL) was applied (50 μL/minute) to a chip matrix activated by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) to achieve 1000 resonance response units (RU). The remaining reactive chemical groups on the chip were blocked by applying Ethanolamine hydrochloride-NaOH for one minute. Next, the following samples were applied to the matrix for two minutes and 30 seconds: a) phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.05% Tween 20; b) canine IL-4 (R&D Systems, Minneapolis, USA) at 125, 250 or 500 ng/mL; c) canine IL-10 (R&D Systems) at 31.2, 62.5, 125, 250 or 500 ng/ml. IL-4 and IL-10 were diluted with PBS containing 0.05% Tween 20. After each analyte binding evaluation, matrix regeneration was performed by applying regeneration buffer for 30 seconds. Each sample was evaluated twice and results are presented as means and standard deviations (X±SD) of RU. Kinetics of association (Kon) and dissociation (Koff), as well as affinity of rcasIL-10R1 to IL-10, were calculated from RU measurements using Origin v.8.5 software (OriginLab Corporation, Northampton, USA).

All the data including growth parameters were statistically analyzed using SAS. For N availability data at the different growth stages, separation of means was subjected to Tukey’s honestly significant difference test using JMP 9.0 (SAS Institute Inc., Cary, NC, United States). The graphing was done using Origin v.8.5 software (Originlab Corporation, Northampton, United States). Correlation analysis was conducted to identify relationships between the measured parameters. All tests were performed at the 0.05 significance level.

For proteins that exhibited changes in concentration as revealed by label free quantitative proteomics, intensity values were pooled with Bio-Plex™ protein concentration data. The protein concentration data were mean centred and autoscaled prior subjection to principal component analysis using the pcamethods script (http://www.bioconductor.org) in R (http://www.R-project.org). For all individual protein species, ANOVA was performed followed by Tukey posthoc analysis (origin v.8.1, originlab, Northampton, MA, USA).