Contribution
Overview
Our project aimed to reduce the virulence of Aeromonas hydrophila, a harmful aquatic pathogen, to protect the aquaculture industry. We introduced AHL lactonases into Bacillus subtilis WB600. The engineered B. subtilis disrupted the quorum sensing of A. hydrophila by degrading AHL (C4-HSL), reducing its biofilm formation and extracellular protease activity. B. subtilis WB600 harboring pHT43-AiiA proved to be the most effective, making it a promising probiotic for aquaculture applications.
The following are our contributions:
1. Developed a Standardized Procedure for Quorum Quenching Research
Through literature research, we organized and developed a standardized procedure for studying AHL-induced quorum quenching for the iGEM community, which includes the following stages:
Stage 1: Direct AHL Deactivating Tests
Use Chromobacterium subtsugae CV026 (for short-chain AHLs) or VIR07 (for long-chain AHLs) as biosensors for detecting AHLs. Use artificial AHL first, then natural AHL from the target organism. Use the agar well diffusion method and measure the width of the purple/colorless rings formed, indicating AHL degradation.
Stage 2: Testing Quorum Sensing-Induced Changes
The effects of quorum sensing vary across different species. For pathogens, the focus is usually on toxin production, biofilm formation, protease activity, and antibiotic resistance. Test each factor by culturing the target organism in media containing the substance of interest.
The detailed Procedure is attached as a PDF for future iGEM teams to use.
2. Characterization of Pgrac
We characterized Pgrac (https://parts.igem.org/Part:BBa_K1628202) for its ability to induce quorum-quenching in Bacillus subtilis against Aeromonas hydrophila. The quorum quenching abilities of Pgrac-controlled AHL lactonases (AiiA, YtnP, AttM, AiiM, AhlD, AhlS) were evaluated through synthetic AHL degradation tests (Figure 1A-B), natural AHL degradation tests (Figure 1C), biofilm reduction tests (Figure 2A-B), and extracellular protease reduction tests (Figure 2C-D). We demonstrated that Pgrac can control the expression of endogenous AHL lactonases in B. subtilis WB600, and showed that introducing all genes except AttM (BsAiiA, BsYtnP, MtAiiM, AsAhlD, and SsAhlS) under the control Pgrac enhanced the C4-HSL degrading ability of B. subtilis WB600. Among all groups, B. subtilis WB600 expressing AiiA under the control of Pgrac is the most effective quorum quenching strain against A. hydrophila.
Figure 1. A-B. Synthetic AHL degradation tests; C. Natural AHL degradation test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Figure 2. A-B. Crystal violet A. hydrophila biofilm assay. C-D. The activity of the extracellular proteases of A. hydrophila. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
3. Developed a Safety Handling Manual for Aeromonas hydrophila
This year, we worked with A. hydrophila, a Risk Group 2 microorganism. For future iGEM teams using this organism, we strongly recommend adhering to the following safety guidelines to ensure safety:
Personal Protective Equipment (PPE): Team members must wear lab coats and gloves, which should be changed after handling the bacteria. Masks are optional but recommended.
Bacteria Storage: A. hydrophila must be stored in clearly labeled, leak-proof containers. These containers must not leave the laboratory under any circumstances.
Handling: Containers should only be opened in a Class II biosafety cabinet within a BSL-2 lab. They must be sealed before removal from the cabinet. After use, the UV light in the biosafety cabinet should be activated to ensure decontamination.
Prohibited Practices: The use of needles, syringes, or sharp objects when handling A. hydrophila is strictly prohibited.
Spill Response: In the event of a spill, cover the area with paper towels and apply disinfectant, allowing sufficient contact time before cleanup.
Decontamination: All materials that have come into contact with the bacteria must be autoclaved before disposal.
Health Monitoring: Team members working with A. hydrophila should monitor their health closely. Any symptoms such as abdominal pain, diarrhea, or vomiting should be reported for medical evaluation.
4. Bacillus subtilis Transformation Troubleshooting
The two main transformation methods for B. subtilis are electroporation and chemical methods. We initially used electroporation, but despite colony growth on selective plates, no positive transformants were detected in colony PCR tests. Adjustments to voltage, plasmid amount, and buffer concentrations yielded no success. Interestingly, B. subtilis developed antibiotic resistance even in negative controls, possibly due to stress-induced tolerance. Due to these issues, we switched to chemical transformation using the Paris method with Spizizen’s minimal medium. Colony PCR confirmed successful transformants, with almost no false positives. This method proved highly efficient and resolved our challenges.
Our experience provides a guide for troubleshooting B. subtilis transformation, offering a strategy that future teams can follow. For more details, see Engineering Success https://2024.igem.wiki/hangzhou-biox/engineering.
5. Clarified the approval process for GM microorganisms in China
We interviewed experts, conducted literature research, and reviewed regulations related to GMOs in China.
General Biosafety
Under the Biosecurity Law of the People's Republic of China (2020), genetically engineered microorganisms undergo strict reviews, including ethics assessments and compliance with the Measures for the Safety Evaluation of Agricultural GMOs (2016), overseen by the Ministry of Agriculture. Containment of modified strains is required to prevent environmental release.
Food Safety
Under the Food Safety Law of the People's Republic of China (2015), GMOs in food must be evaluated by the National Center for Food Safety Risk Assessment, comply with the Administrative Measures for New Food Raw Materials (2017), pass evaluations for production licenses, and be clearly labeled.
Environmental Protection
The Environmental Protection Law of the People's Republic of China (2009) mandates ecological impact assessments for GMOs and the use of containment measures like closed systems to prevent contamination.
For more details, visit Human Practices https://2024.igem.wiki/hangzhou-biox/human-practices. We hope our work offers future teams insights into gaining approval for their GM products from Chinese authorities.