To accurately determine the protein concentration of our enzyme extracts, we adopted and optimized the Bradford assay based on the G250 dye-binding method [1]. This colorimetric assay forms a blue complex with basic and aromatic amino acid residues, allowing rapid and sensitive quantification of proteins using minimal equipment. By optimizing parameters such as the dye-to-sample ratio, we minimized inter-sample variation and increased detection sensitivity for crude lysate samples. Using the method, we established a calibration curve using Bovine Serum Albumin(BSA) and reached a highest R² value of 0.9983. After multiple rounds of improvements, we enhanced the precision and reproducibility of our protein quantification pipeline. By ensuring reliable protein concentration data, we minimized potential errors in activity assays, leading to more trustworthy comparisons of enzyme activities. This improved approach provides future iGEM teams with a scalable, accessible solution for accurate protein quantification, making it easier to analyze enzyme performance.

To determine the concentration of protein in a sample by measuring the color change when Coomassie Brilliant Blue dye binds to ptotein.

The Bradford assay quantifies protein concentration by measuring the binding of Coomassie Brilliant Blue G-250 dye to proteins. In an acidic solution, the dye shifts from a reddish-brown color to blue upon binding primarily to basic and aromatic amino acid residues in proteins. This color change results in a measurable increase in absorbance at 595 nm. The intensity of the blue color is directly proportional to the protein concentration, allowing for quantification by comparison to a standard curve generated with known concentrations of a standard protein like Bovine Serum Albumin (BSA).

To evaluate the enzymatic potential of our chitinase and glucanase candidates, we employed the DNS assay to measure reducing sugar release from substrate hydrolysis [2]. This colorimetric method provides a low-cost and high-throughput approach using only 1.5mL centrifuge tubes, 96-well plate and dry bath, enabling rapid screening of multiple candidates and substrates. We applied this assay to substrates relevant to fungal cell walls, including colloidal chitin, laminaran, lichenan, and pustulan. By optimizing reaction conditions for each enzyme, we quantified specific activities (U/mg) based on protein concentration determined via Bradford assay (Coomassie Brilliant Blue G-250). This strategy allowed us to efficiently compare catalytic efficiencies and substrate preferences across our enzyme library, identifying promising candidates for further antifungal applications. Through this approach, we not only systematically compared the catalytic efficiency and substrate specificity of our enzyme candidates, but also established a standardized, cost-effective framework for high-throughput enzyme screening. This provides a quantitative foundation for rational enzyme selection in a broad range of applications and offers a generalizable methodology for future iGEM teams developing enzyme-based biocatalytic systems.

To quantify the enzymatic activity of chitinase and glucanase candidates by measuring the release of reducing sugars using the DNS method.

The 3,5-Dinitrosalicylic acid (DNS) assay quantifies the concentration of reducing sugars. Under alkaline and high-temperature conditions, the DNS reagent reacts with the free carbonyl group of reducing sugars (e.g., glucose). This reaction reduces the yellow-colored DNS to 3-amino-5-nitrosalicylic acid, which is orange-red. The intensity of this orange-red color is directly proportional to the concentration of reducing sugars present. The absorbance is measured at 540 nm, and the reducing sugar concentration of an unknown sample is determined by comparing its absorbance to a standard curve generated with known concentrations of a reducing sugar (like glucose).

X = (n · c · 1000)/(t · M)

where X is the enzyme activity of the sample (U/mL), n is the dilution factor, c is the glucose concentration (mg/mL) calculated from the glucose standard curve regression equation, t is the reaction time (min), and M is the molecular weight of reducing sugar (glucose, 180.16 g/mol; N-acetyl-glucosamine, 221.21 g/mol).

Specific Activity (U/mg) = Enzyme Activity (U) / Enzyme Mass (mg) (Protein mass determined via Bradford assay.)

Through extensive literature research and experimental validation, we constructed a comprehensive antifungal gene collection consisting of chitinase, glucanase, human lysozyme, and geraniol biosynthetic genes, targeting the structural polysaccharides and membranes of fungal cells. To improve expression and activity, certain enzymes were computationally optimized. Fusion with carbohydrate-binding modules (CBMs) for surface immobilization was also attempted. Each part was functionally verified via protein expression, enzymatic assays, and antifungal activity evaluation. These contributions expand the iGEM Parts Registry with a modular, experimentally validated toolkit for developing bio-based antifungal systems and enzyme optimization studies.

We established a stable fermentation system and a GC-MS measurement method for geraniol production. This protocol was developed for geraniol and can be adapted by other teams working on geraniol fermentation. By sharing the detailed protocol with the community, we aim to support broader efforts in monoterpene fermentation and encourage further method improvements. The following is the protocol of this fermentation method with detailed steps:

In the GC-MS analysis, Column is Agilent J&W DB-5ms Ultra Inert (Ul),30 m x 0.25 mm 0.25 um. The split ratio was set at 5:1, injection port temperature 70°C. GC oven temperature was initially held at 50°C for 2 min, ramped with a gradient of 96°C/min to 300°C and held for 2 min. Using this method, we are able to plot a standard curve and obtain the accurate concentration of geraniol produced. (Fig. 4)

The existing part BBa_K2380005, encoding chitinase A1 from Bacillus circulans (BcChiA1), possesses high efficiency and specificity towards hydrolysis of β-1,4-N-acetylglucosamine polymers. To expand its applicability and provide functional data, we carried out experiments with this part under a standardized protein expression and enzymatic testing framework (Fig. 5a). Through SDS-PAGE, we first verified that BcChiA1 can be stably and solubly expressed in E. coli BL21(DE3) (Fig. 5b). Quantitative analysis using ImageJ and Bradford Method (Coomassie Brilliant Blue G-250) are utilized to test the accurate quantity of protein expressed (Fig. 5c). To assess enzymatic performance, we conducted DNS assay to quantify BcChiA1 activity under a single condition and over time (Fig. 5d). The results demonstrated that BcChiA1 exhibits significant hydrolytic activity towards colloidal chitin. The glucose concentration rises sharply in the first 20 min, and almost reach the plateau in the first 40 min (Fig. 5e).

Building upon this, we introduced BaCBM2e fusion constructs to enable enzyme immobilization to materials on various surfaces (Fig. 6a). These constructs were verified by DNA gel electrophoresis, showing successful construction of targeted plasmid through Gibson Assembly (Fig. 6b). BaCBM2e-BcChiA1 was successfully expressed and tested with DNS assay, and the results show increased activity, demonstrating that adding an additional BaCBM2e domain also increases the affinity of the original chitinase towards its substrate, chitin (Fig. 6c,d).

Collectively, these experiments not only deepen the functional understanding of BBa_K2380005 but also extend its utility in real-world applications such as surface-immobilized antifungal enzyme systems. All protocols, plasmid maps, and raw data are openly documented to support further optimization by future iGEM teams.