Existing Cereulide Detection Methods VS Our Test Kit

      Currently, there are two main methods to detect cereulide: fluorescent probes[1] and conventional high-performance liquid chromatography (HPLC) connected to a tandem mass spectrometer (LC-MS/MS)[2].

Existing Methods Strength Weakness
Fluorescent Probes
  • Convenient
  • Portable
    		•
  • Fluorescence titration of the sample and a benchtop fluorometer are required.
  • Require laboratory skills.
    		•
    LC-MS/MS
  • High sensitivity
  • High specificity
    		•
  • Expensive, typically between $100 and $200 per sample, in terms of machine acquisition, daily operation, and routine maintenance.
  • Long time for sample preparation.
  • Require well-equipped laboratory.
    		•

          Regarding the limitation of current detection methods, our team aims to provide a cost-effective and user-friendly method for detecting cereulide, specifically targeting food suppliers and food service company users without laboratory backgrounds.

    Overview of Our Hardware

          We have engineered B. Subtilis cells as biosensors for cereulide detection, taking advantage of the biological motifs of our chassis, Bacillus Subtilis.

          To enhance the effective diffusion of hydrophobic cereulide into our biosensor cells and eliminate the need for liquid cell culture handling, we have adopted a PVA-PEG gels-based whole-cell biosensor approach.

    Figure 1 B. Subtilis Whole-Cell Biosensor Working Principle

          We embed the sporulated B. Subtilis cells within PVA-PEG gels and form solid discs allowing users to simply activate the cells and add rice-extracted samples onto gels to obtain the detection results. Additionally, PVA(Polyvinyl Alcohol) and PEG(Polyethylene Glycol) powders are inexpensive, and the process of making PVA-cells gels is easy for general laboratories, as it only requires heating for polymerization.

    Why use PVA Hydrogel as a biosensor platform?

          There are two main reasons, cell immobilization for detection limits optimization, as well as cell immobilization to ensure safety and user-friendliness.

    Figure 2 Cell Immobilization in Hydrogel

          Cereulide is hydrophobic, it would be difficult for it to diffuse and reach cells in a liquid medium and trigger circuits to give signals. Based on our research, PVA hydrogel-based cell biosensors have been applied by researchers to detect hydrophobic toxins. For instance, a team had developed luminescent yeast cells entrapped in hydrogels for hydrophobic chemical biodetection[3] Another proof of concept mentioned in literature[4] is that cell immobilization could improve the sensor assembly by increasing mechanical and chemical stability, facilitating close contact between the medium and analyte, thus minimizing interference, resulting in improved detection limits. We carried out experiments to successfully prove these concepts.

          Although B. subtilis is a BSL Level 1 bacterium and is considered safe for human consumption as probiotics, concerns arise regarding liquid handling and direct contact with bacteria. User safety is a top priority based on feedback from our survey respondents. To address this, we transformed the liquid biosensor into a solid one. Additionally, we consulted Professor Marshal Liu, an expert in food technology and bioproducts from HKUST Chemical and Biological Engineering, to ensure the safety of our test kit design.

    Figure 3 Questionnaire results with 119 responses received

         

         

    Why Our Test Kit?

    Safe

    Top prioritize by questionnaire respondents
  • Utilizing B. subtilis, a bacterium classified as BSL 1
  • Prevent leakage and avoid liquid bacteria culture handling
  • Cell culture immobilization within the packaging
  • Cell-gel trapping by the package
  • Killing agents: Home-based bleach could be employed to kill engineered bacteria embedded in gel effectively[5]

    • Reliable

    2nd top prioritize by questionnaire respondents
  • Specific to Cereulide
  • Sensitive: Able to test the minimal level of 3 μM cereulide analog, valinomycin
  • Higher Fluorescence protein viability in Hydrogel compared to LB medium

    • User-friendly

  • Low user barrier: No lab technique required
  • Avoidance of liquid bacterial culture handling techniques
  • No bulky hardware installation
  • Easy sampling for testing: Convenient sample collection process

    • Cost-effective

  • From our questionnaire result, we found that the acceptable price range of a test kit is $15-45 (add up is 62.1%)
  • Our Estimated cost of package is $35.616 HKD < $45 HKD (See our package)

    • Easy construction for laboratory production

  • B. Subtilis Spores embedded in gel could be used within 5 days after construction and storage at 4°C.
  • Storability (sporulation and germination ability of B. subtilis): Capability for long-term storage/shipping stability through sporulation.

    • Hardware Contribution

    Novelty

  • We are the first iGEM project that successfully uses PVA Hydrogel for B. subtilis whole-cell biosensor construction for higher effectiveness of hydrophobic toxin testing.

    • Potential Implementation

  • Our hardware design, PVA Hydrogel, can help organisms grow according to experimental parameters and execute their engineered function.
  • Future iGEM teams could construct PVA Hydrogel as a new option for whole-cell biosensors, especially if they are testing hydrophobic toxins.

    • Future Improvement

    Limitations & Challenges Future Plans
    Insufficient time to complete testing with actual cereulide. Testing with Cereulide apart from its analog to assess circuit functionality.
    Limited number of cell-gels for imaging and quantitative analysis + Inadequate image database for training.
  • Increase production of cell-gels to expand the image database and enable more comprehensive training for quantitative analysis.
  • Relate the photos taken from Blue Light Transilluminators and Fluorescence intensity figures measured by plate readers, and establish a correlation between the visual observations and the quantitative data.
    		•
    Insufficient fluorescence intensity upon Valinomycin/Cereulide addition in cells-gel assay. Go through further engineering cycle of the cell, for instance, add a forward feedback loop, to amplify the fluorescence production efficiency.
    Insufficient fluorescence intensity upon Valinomycin/Cereulide addition in spores-gel assay. Modify the protocol of Spores and Spore Gel by longer germination time and do further testing.
    Extraction methods for Cereulide from rice samples based on research papers. Access to LCMS for quantification of Cereulide yield after different extraction methods for further validation.

    References

      [1] J. García-Calvo et al., “Potassium-Ion-Selective Fluorescent Sensors To Detect Cereulide, the Emetic Toxin of B. cereus, in Food Samples and HeLa Cells,” ChemistryOpen (Weinheim), vol. 6, no. 4, pp. 562-570, 2017, doi: 10.1002/open.201700057.
      [2] P. H. in 't Veld, L. F. J. van der Laak, M. van Zon, and E. G. Biesta-Peters, “Elaboration and validation of the method for the quantification of the emetic toxin of Bacillus cereus as described in EN-ISO 18465 - Microbiology of the food chain - Quantitative determination of emetic toxin (cereulide) using LC-MS/MS,” International journal of food microbiology, vol. 288, pp. 91-96, 2019, doi: 10.1016/j.ijfoodmicro.2018.03.021.
      [3] T. Fine et al., “Luminescent yeast cells entrapped in hydrogels for estrogenic endocrine disrupting chemical biodetection,” Biosensors & bioelectronics, vol. 21, no. 12, pp. 2263-2269, 2006, doi: 10.1016/j.bios.2005.11.004.
      [4] E. Wahid, O. B. Ocheja, E. Marsili, C. Guaragnella, and N. Guaragnella, “Biological and technical challenges for implementation of yeast-based biosensors,” Microbial biotechnology, vol. 16, no. 1, pp. 54-66, 2023, doi: 10.1111/1751-7915.14183.
      [5] K. Schulz-SchönHagen, "Bacillus subtilis biosensors: Engineering a living material sensor platform," Ph.D. dissertation, ETH Zurich, 2019. [Online]. Available: https://www.research-collection.ethz.ch/bitstream/handle/20.500.11850/389191/2/eth-26452-02.pdf.