Our Project

Our project is dedicated to providing a convenient, efficient, and environmentally friendly method for the detection and degradation of OTA in wine. Traditional detection methods often rely on the use of large-scale instrumentation, incurring higher costs, and some involve the utilization of environmentally detrimental materials. Additionally, existing degradation methods face cost-related challenges, and the application of certain physical and chemical techniques may potentially compromise the sensory attributes of the wine. In light of these limitations, we have employed synthetic biology methodologies to engineer a cell-free detection module and a degradation module, facilitating the precise identification and remediation of OTA in wine.

Our Final Product

Our team's final product consists of two components: an OTA detection module and a degradation module. The detection module features pre-fabricated reaction systems with adaptamer and dumbbell template dry powders, among others. To use it, water, samples, and the necessary enzymes are added, and the reaction occurs within a fluorescence quantitative PCR instrument.In contrast, the degradation module utilizes engineered bacteria encapsulated in sodium alginate and chitosan matrices. To activate it, these engineered bacteria are simply introduced into pre-prepared sodium alginate beads.Crucially, extensive laboratory testing has validated the consistent and dependable performance of our device.

Device

Our product consists of two components: the cell-free detection module and the degradation module.

Cell-Free Detection Module: This module describes a detection reagent kit that includes adapter primer dry powder, dumbbell template dry powder, magnetic beads (MBs), dilution solvent (ddH₂O), and PCR reaction tubes, all meticulously designed to ensure optimal performance.

Degradation Module: This module includes engineered bacteria encapsulated with sodium alginate and chitosan.As illustrated in figure 1, the engineered bacteria are situated within the innermost layer, enclosed by a sodium alginate matrix. The outermost layer comprises chitosan, providing structural reinforcement to the sodium alginate encapsulation.

Fig. 1 immobilized microcapsules for Encapsulation of Engineered E. coli

Steps

Cell-Free Detection Module: Firstly, the adapter primer dry powder and dumbbell template dry powder are mixed in the appropriate proportions to formulate the reaction system, into which the sample is introduced. Under the conditions of ochratoxin A presence, rolling circle amplification is carried out using a fluorescence quantitative PCR instrument, resulting in the amplification and subsequent detection of fluorescence signals. Operators can determine if the OTA level is below the standard by analyzing the fluorescence intensity.

Degradation Module: Engineered bacteria encapsulated with chitosan and sodium alginate will be immobilized at the inlet of the raw red wine stream. As the red wine flows through, these engineered bacteria will engage in a reaction to degrade ochratoxin A.

Storage

Cell-Free Detection Module: Storage at 4℃ in Refrigerator.

Degradation Module: Storage at -20℃ in Refrigerator.

For Products

Our detection module incorporates lyophilized powders, presenting a potential risk of biological contamination if they leak into the environment. Laboratories or processing facilities employing this assay kit should meticulously follow the provided operating instructions and delegate waste disposal to specialized organizations.

In our degradation module, the core components consist of engineered bacteria enclosed within sodium alginate and chitosan, forming sodium alginate particles. In the event of a rupture in these particles' casing, the enclosed substances may potentially diffuse into the wine, adversely impacting the quality of red wine and even posing a risk of biological contamination. Hence, it is essential for facilities to guarantee the reliable immobilization of sodium alginate particles and their prompt replacement when necessary.

For Experiment

Throughout our experiments, strict adherence to iGEM's security policy was maintained. All validation procedures were conducted exclusively within the laboratory environment, with no release of engineered bacteria into the external surroundings.

During the course of our experimental procedures, we also employed lyophilized powder and rigorously adhered to regulations to prevent any environmental contamination. In the validation of our product's feasibility, we utilized ochratoxin A, which is a toxic substance. We meticulously followed experimental protocols, implemented necessary protective measures, and safely managed its disposal to ensure the security of both the laboratory and the environment.

Application of our kit

1. Compared to conventional physical detection methods relying on large-scale equipment, our adapter-dependent cell-free detection technology still exhibits some sensitivity gaps. Further system optimization is necessary to enhance detection accuracy.

2. While our degradation methods have proven effective in laboratory conditions, their stability and performance under complex environmental conditions in real-world industrial production settings require further validation and potential adjustments.

3. Red wine, being a beverage, demands exceptional flavor standards. Our degraded product has been confirmed to meet safety standards, and theoretically, its impact on the quality of red wine is minimal. However, further testing is required to assess the extent of its influence on the wine's flavor.