This year, our team aimed to engineer Lactobacillus reuteri, a Generally Recognized as Safe (GRAS) probiotic, to secrete polygalacturonase (pgxC) for efficient fruit peel degradation. Throughout our Engineering Cycle (please see our Engineering page for more information), we identified several quantitative experiments that required systematic measurement:
Bradford Assay: Confirming the total protein secretion levels in engineered L. reuteri containing the pgxC construct on the shuttle vector pTRKH3, in comparison with control strains harboring an empty plasmid.
SDS-PAGE Analysis: Assessing the presence of the expected 48.5 kDa pgxC protein band in the supernatant, confirming successful secretion via the Usp45 signal peptide.
DNS Assay (Enzyme Activity Assay): Quantifying the release of D-galacturonic acid from pectin as a direct measure of pgxC enzymatic activity in degrading fruit peel polysaccharides.
The Bradford assay was essential during the ‘Build’ phase of our Engineering Cycle, providing quantitative data on protein secretion levels. SDS-PAGE further validated the molecular size and integrity of the expressed enzyme. Finally, the DNS assay was critical during the ‘Test’ phase, as it directly measured enzymatic activity and functional pectin degradation.
However, we encountered two key limitations. First, both the Bradford and DNS assays required precise standard curves (BSA for protein, D-galacturonic acid for reducing sugar quantification) to ensure reliable normalization and comparability. Second, variability in fruit peel composition necessitated standardized substrate preparation to minimize experimental bias. Accordingly, our measurement objectives for this year were the following:
Bradford Assay: Establishing a reliable standard curve to quantify total protein secretion in engineered vs control L. reuteri cultures.
SDS-PAGE: Verifying the presence of the secreted pgxC protein (48.5 kDa) and distinguishing it from background proteins.
DNS Assay: Generating a standard curve with D-galacturonic acid to accurately quantify enzymatic activity, measured as µmol of reducing sugars released per mL per minute.
Measurement Part 1: Bradford Assay
Measurement Background
The Bradford assay, developed by Marion M. Bradford in 1976, is a widely used biochemical assay for quantifying protein concentration in a solution. It is colorimetric and relies on a dye’s absorbance shift when binding to proteins. In this project, the Bradford assay was applied to measure total protein secretion from engineered Lactobacillus reuteri carrying the pgxC construct. This allowed us to compare protein output between engineered strains and plasmid-free control strains to confirm enhanced secretion of pgxC.
Measurement Principle
(Figure 1. Principle of Bradford assay.)
The assay is based on the interaction between Coomassie Brilliant Blue G-250 dye and proteins in an acidic solution. Binding occurs mainly at positively charged amino acid residues (arginine, lysine, histidine), which induces a shift in absorbance from 465 nm (brown) to 595 nm (blue). The intensity of the blue color is proportional to protein concentration. By preparing a BSA (bovine serum albumin) standard curve, we could quantitatively determine the protein concentrations secreted into the culture supernatant of engineered vs control L. reuteri.
Measurement Protocols
(Figure 2. Protocol of Bradford protein assay.)
[Materials]
Culture supernatants from engineered L. reuteri (pgxC-expressing) and control strains
Bovine serum albumin (BSA) standard solution (2 mg/mL)
Bradford reagent
96-well clear, flat-bottom microplate
Microplate reader (set to 595 nm)
Micropipettes and sterile tips
Distilled water
[Procedures]
Step 1. Prepare BSA Standard Curve
Prepare six BSA standards in 1.5 mL tubes by diluting 2 mg/mL BSA stock with distilled water:
(Table 1. Preparation of BSA standard solutions for Bradford assay calibration curve.)
Step 2. Prepare Diluted Bradford Reagent
Dilute Bio-Rad Bradford reagent 1:5 with distilled water:
Step 3. Set Up 96-Well Plate
Add 195 µL of diluted Bradford reagent into each well.
Add 5 µL of each BSA standard and 5 µL of unknown culture supernatant samples.
Set your microplate reader to measure absorbance at 595 nm.
Zero the microplate reader using the blank well containing distilled water.
Measure the absorbance of standard solution and protein samples in the microplate.
Step 5. Data Analysis
Plot absorbance (y-axis) vs BSA concentration (x-axis).
Fit a linear regression line and record R² value.
Use the regression equation to calculate protein concentrations of unknown samples.
Report concentrations as µg/µL (normalized against culture OD600 for consistency).
<Measurement Results>
(Figure 3. The BSA standard curve was used for quantifying the proteins presented in cell culture media.)
The following trendline equation was derived from the BSA standard curve (example values):
(Figure 4. The BSA standard curve equation for calculating protein concentration.)(Table 2. Protein concentration was calculated based on the absorbance.)(Figure 5. Quantification of secreted protein concentration in control and engineered L. reuteri. Protein levels in culture supernatants were measured using the Bradford assay. Engineered strains expressing pgxC secreted significantly higher protein concentrations (~0.10 µg/µL) compared to control strains containing the empty plasmid (~0.04 µg/µL). Data represent mean ± SD from three biological replicates. Statistical significance was assessed using a two-tailed Student’s t-test (p < 0.05).)
The results demonstrated that engineered strains secreted 2.5-fold more total protein than the plasmid-free controls. This outcome is consistent with the successful expression and secretion of the pgxC enzyme (48.5 kDa), supported by SDS-PAGE band visualization (Figure 6).
(Figure 6. Detection of secreted pgxC protein in L. reuteri. (Left) Illustration showing separation of cell pellet and supernatant, with pgxC proteins detected in the extracellular fraction. (Right) SDS-PAGE gel analysis of culture supernatant (S) and cell lysate (L) from L. reuteri strains with and without plasmid expression. A distinct protein band corresponding to ~48.5 kDa, consistent with the predicted size of pgxC, was observed in the supernatant and lysate of cells harboring the pgxC plasmid, but not in controls lacking the plasmid.)
Measurement Discussion
Prior to the Bradford assay, we lacked a direct normalization reference for pgxC secretion in L. reuteri. However, by utilizing bovine serum albumin (BSA) as a normalization standard, we were able to generate a least-squares regression line predicting absorbance at 595 nm for different protein concentrations. The regression analysis produced the equation shown in Figure 4.
This strong positive linear correlation validated the reliability of using the BSA calibration curve to estimate total protein concentrations. This allowed us to compare protein secretion between engineered L. reuteri strains carrying the pgxC construct and plasmid-free control strains. Specifically, we described the process of (1) constructing a BSA standard curve and (2) applying the regression line to predict protein concentrations in experimental samples (Figures 4-5).
To obtain a calibrated regression line, we included a negative control (0 µg/µL BSA). The x-axis represented BSA concentration (µg/µL), while the y-axis represented absorbance values (a.u.). The regression line was fitted using least-squares analysis and exhibited strong linearity across the tested concentration range. We recommend that future iGEM teams adopt similar calibration measures to ensure accuracy and reproducibility when quantifying protein concentrations from microbial supernatants.
Nonetheless, several limitations were identified. First, the Bradford assay measures total protein content, not the specific amount of pgxC enzyme. This means that secreted host proteins and background peptides could contribute to the absorbance signal, potentially overestimating pgxC yield. Second, variability in secretion efficiency between replicates may introduce noise into the dataset. While the SDS-PAGE confirmed the presence of a distinct 48.5 kDa pgxC band, quantification from Bradford alone cannot distinguish between pgxC and others (Figure 6).
Therefore, the Bradford assay served primarily as a supportive normalization tool, complementing SDS-PAGE and DNS activity assays. By combining protein quantification with enzyme activity measurements, we ensured that increases in total protein levels corresponded with actual increases in functional pgxC activity. This layered approach enhances measurement accuracy and provides a framework that future teams can adopt when working with secreted proteins in probiotic chassis.
Future Suggestions for iGEM Teams
(Figure 7. Future suggestions for future iGEM teams for the BSA assay.)
For future iGEM teams using the Bradford assay, we recommend several strategies to improve accuracy and interpretability. First, including multiple independent protein standards (such as BSA, lysozyme, and casein) can help account for amino acid composition biases, since Bradford dye binding is highly influenced by arginine and aromatic residues. Second, normalizing protein concentration values to cell density (OD600) or dry cell weight will make cross-experiment comparisons more robust, especially when secretion yields differ with growth conditions. Third, combining Bradford assay results with Western blotting or ELISA against the specific enzyme of interest would allow teams to distinguish pgxC from background host proteins, providing greater confidence in quantification. Finally, adopting triplicate technical repeats and biological replicates, and reporting R² values for calibration curves, will ensure reproducibility and transparency. These practices will allow future teams to build on Bradford assay data as a reliable normalization tool within integrated measurement frameworks.
Measurement Part 2: DNS Assay
Measurement Background
The 3,5-dinitrosalicylic acid (DNS) assay is a colorimetric method widely used to measure the activity of glycoside hydrolases, including polygalacturonase (pgxC). This assay quantifies the reducing sugars released when enzymes degrade polysaccharide substrates, such as pectin. In this project, the DNS assay was employed to evaluate the enzymatic activity of secreted pgxC from engineered L. reuteri by detecting D-galacturonic acid released from pectin substrates.
Measurement Principle
DNS reagent reacts with reducing sugars (such as D-galacturonic acid) under alkaline and high-temperature conditions, producing a reddish-brown complex.
The absorbance of this complex is measured at 540 nm using a spectrophotometer.
The intensity of the color is proportional to the concentration of reducing sugars.
A D-galacturonic acid standard curve allows conversion of absorbance values to µmol of reducing sugars released.
Measurement Protocols
(Figure 8. Overview of the DNS assay workflow. Pectin solution is incubated with culture supernatant at 37 °C to release reducing sugars, the reaction is stopped by adding DNS reagent, and tubes are heated in a boiling water bath for 5 min to develop the reddish-brown chromophore. After cooling, absorbance is measured at 540 nm and reducing sugars are quantified against a D-galacturonic acid standard curve (0–1.0 mg mL⁻¹). Blanks (substrate + DNS without enzyme) and biological triplicates are included for each condition.)
[Materials]
DNS reagent (3,5-dinitrosalicylic acid, NaOH, potassium sodium tartrate)
Pectin substrate solution (1% w/v)
D-galacturonic acid standard (for calibration curve)
Culture supernatants from engineered and control L. reuteri strains
Add 0.5 mL of each standard to 0.5 mL DNS reagent.
Heat samples in a boiling water bath for 5 minutes.
Cool to room temperature, then measure absorbance at 540 nm.
Plot absorbance vs concentration to generate standard curve.
Step 2: Sample Preparation
Mix 0.5 mL of pectin substrate (1% solution) with 0.5 mL of culture supernatant (engineered or control strain).
Incubate at 37 °C for 30 minutes.
Add 0.5 mL DNS reagent to stop the reaction.
Heat in boiling water bath for 5 minutes, cool, then measure absorbance at 540 nm.
Step 3: Data Analysis
Use the D-galacturonic acid standard curve to calculate reducing sugar concentration.
Express enzyme activity as µmol D-galacturonic acid released per mL per minute (U/mL).
Measurement Results
(Figure 9. Standard curve for D-galacturonic acid quantification using the DNS assay. The standard curve was generated by measuring absorbance at 540 nm for increasing concentrations of D-galacturonic acid (0–1.0 mg/mL). The linear regression equation (A = 1.207·C + 0.016) with an R² value of 1.000 indicated excellent linearity across the tested range. This curve was subsequently used to calculate the concentration of reducing sugars released after incubation of pectin with pgxC-secreted proteins from Lactobacillus reuteri.)(Figure 10. Fruit peel samples prepared for DNS assay analysis. Homogenized fruit peel extracts (tomato, pear, watermelon, kiwi, and banana) were collected and processed. DNS-based colorimetric reactions were performed in a 96-well plate, showing visible color development corresponding to reducing sugar release from different peel substrates.)(Figure 11. Quantification of D-galacturonic acid after 0, 12, 24, 48h incubation of supernatant produced from control (no pgxC) and engineered (pgxC) samples. Values are shown as mean ± SD from three replicates.)
To evaluate pgxC activity, culture supernatants from control (empty plasmid) and engineered L. reuteri strains were incubated with pectin for 0, 12, 24, and 48 hours. The DNS assay revealed a time-dependent increase in reducing sugar concentration in the engineered strain, whereas control samples showed minimal activity:
(Table 3. DNS assay quantification of reducing sugars released from pectin degradation.)(Figure 12. Quantification of D-galacturonic acid release from pectin degradation using the DNS assay. Culture supernatants from control (empty plasmid) and engineered (pgxC-expressing) Lactobacillus reuteri were incubated with pectin for 0–48 h. Reducing sugar release was quantified by DNS assay and expressed as D-galacturonic acid concentration (mg/mL) using a standard curve. Engineered strains exhibited a significant time-dependent increase in reducing sugar production (~0.28 mg/mL at 12 h, ~0.49 mg/mL at 24 h, and ~0.54 mg/mL at 48 h), whereas control strains showed negligible activity. Data represent mean ± SD of three replicates. Statistical significance was assessed using a two-tailed Student’s t-test (p < 0.05, p < 0.01).)
Figure 10 shows the time-course quantification of D-galacturonic acid released from pectin degradation by culture supernatants of control and engineered Lactobacillus reuteri. At the 0 h time point, both control and engineered strains displayed negligible absorbance at 540 nm, corresponding to very low concentrations of reducing sugars (~0.04 mg/mL in the engineered strain). By 12 h, the engineered strain exhibited a substantial increase in reducing sugar release (~0.28 mg/mL), significantly higher than the control strain (~0.02 mg/mL) (p < 0.05).
At 24 h, the engineered strain continued to accumulate D-galacturonic acid, reaching ~0.49 mg/mL, while the control strain remained unchanged (~0.03 mg/mL). The difference between the two groups was highly significant (*p < 0.01). By 48 h, the engineered strain achieved the highest yield of ~0.54 mg/mL D-galacturonic acid, whereas the control remained below 0.05 mg/mL. This confirmed that the engineered strain maintained active enzymatic degradation of pectin over extended incubation, while the control lacked significant activity (*p < 0.01).
Together, these results demonstrate that pgxC-expressing L. reuteri secreted functionally active polygalacturonase capable of hydrolyzing pectin into D-galacturonic acid in a time-dependent manner. The minimal reducing sugar release in controls validated that the observed activity was specifically due to pgxC secretion rather than background host metabolism.
(Figure 13. Correlation between total protein secretion (Bradford assay) and enzymatic activity (DNS assay) in control vs engineered L. reuteri.)
This integrated comparison highlights the link between protein secretion and enzymatic function in our engineered system. Engineered L. reuteri strains showed ~2.5-fold higher protein secretion compared to plasmid-free controls, as quantified by Bradford assay. Importantly, this increase in total protein correlated with a ~13-fold higher release of D-galacturonic acid from pectin degradation after 48 h, as measured by DNS assay. The correlation strongly supports that the secreted protein was functionally active pgxC polygalacturonase rather than background host proteins. Together, these data validate that engineering L. reuteri to secrete pgxC successfully enhanced both protein yield and functional activity in fruit peel degradation.
Measurement Discussion
The DNS assay was employed to evaluate the enzymatic activity of secreted pgxC from engineered Lactobacillus reuteri. By using D-galacturonic acid as a calibration standard, we generated a linear regression curve:
This correlation confirmed the robustness of the DNS assay in quantifying reducing sugars released during pectin hydrolysis. The calibration allowed us to translate absorbance readings into concentrations of D-galacturonic acid, which reflects the extent of pectin degradation.
Using this method, we compared control strains (plasmid-free) and engineered strains (pgxC-expressing) over a 48-hour incubation period. The engineered strain exhibited a marked time-dependent increase in absorbance at 540 nm, corresponding to reducing sugar concentrations of ~0.28 mg/mL at 12 h, ~0.49 mg/mL at 24 h, and ~0.54 mg/mL at 48 h. In contrast, control strains showed negligible activity, with absorbance values remaining below 0.1 throughout the assay. These findings confirmed that pgxC secretion conferred functional enzymatic activity capable of depolymerizing fruit peel pectin into monosaccharides.
Despite the clear results, several limitations were identified. First, the DNS assay detects total reducing sugars, not exclusively D-galacturonic acid. Thus, other reducing by-products from microbial metabolism may contribute to absorbance, potentially overestimating pgxC-specific activity. Second, the assay requires heating at 100 °C, which can introduce variability if incubation times or cooling rates are inconsistent. Third, the endpoint measurement provides cumulative reducing sugar release but does not capture intermediate oligosaccharides generated during pectin hydrolysis.
To overcome these limitations, we complemented DNS data with Bradford assay measurements of protein secretion and SDS-PAGE confirmation of pgxC expression. Importantly, Figure 11 integrated both assays, directly comparing protein secretion (Bradford) with enzymatic activity (DNS). This combined analysis revealed that engineered strains secreted ~2.5-fold more protein and exhibited ~13-fold higher enzymatic activity compared to controls. The correlation between protein yield and functional activity strongly supports that the secreted protein was pgxC, and not background host proteins.
Overall, the DNS assay provided functional validation of our engineered system by linking molecular measurements (pgxC expression and secretion) to biological function (pectin degradation). When combined with Bradford and SDS-PAGE data, these results confirmed the success of our design and demonstrated the importance of using layered measurement strategies. This integrated approach provides a valuable framework for future iGEM teams aiming to characterize extracellular enzyme activity in microbial chassis.
Future Suggestions for iGEM Teams
(Figure 14. Future suggestions for future iGEM teams for the DNS assay.)
For future iGEM teams planning to apply the DNS assay, we recommend several refinements to increase accuracy and interpretability. First, coupling the DNS assay with HPLC or LC–MS analysis would allow direct quantification of D-galacturonic acid versus other reducing sugars, minimizing background interference. Second, implementing a time-course kinetic assay with multiple shorter intervals (every 2–4 h) could provide a clearer profile of enzymatic activity and substrate depletion. Third, standardizing fruit peel substrate preparation—such as particle size, pH adjustment, and storage conditions—will help reduce variability between biological replicates. Finally, we suggest integrating DNS data with normalization to biomass or OD600 to account for culture growth differences, enabling more rigorous comparison across strains. These improvements would help future teams establish DNS assay results as a quantitative benchmark for characterizing extracellular enzyme activity.
References
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. (SDS-PAGE reference)
Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428. (DNS assay reference)
Compton, S. J., & Jones, C. G. (1985). Mechanism of dye response and interference in the Bradford protein assay. Analytical Biochemistry, 151(2), 369–374. (assay limitations/interferences)
Mohnen, D. (2008). Pectin structure and biosynthesis. Current Opinion in Plant Biology, 11(3), 266–277. (justifies D-galacturonic acid standard for pectin degradation)
