Human Practices

3. Technical Viability Check

Through our literature research, we found that A. hydrophila’s virulence is regulated by quorum sensing via acyl-homoserine lactone (AHL), particularly N-butanoyl-L-homoserine lactone (C4-HSL). When C4-HSL reaches a threshold, it triggers genes that produce toxins, extracellular proteases, and biofilms, facilitating infection (Coquant et al., 2020; Garde et al., 2010; Khajanchi et al., 2009). Disrupting this process through quorum quenching can reduce the bacterium's infectivity (Chen et al., 2013; Chu et al., 2014). AHL lactonases, enzymes that degrade C4-HSL, are naturally abundant and sourced from various bacteria (Dong et al., 2002; Uroz et al., 2008). We believe we can employ these enzymes to weaken A. hydrophila.

 

To evaluate the technical feasibility of our idea of using quorum quenching and gain further guidance, we interviewed Prof. Tianjiang Chu from Zhejiang Ocean University, an aquaculture expert. He restated the importance of controlling A. hydrophila in aquaculture systems and supported our approach of using quorum quenching as a viable solution, boosting our confidence in its effectiveness. Prof. Chu also suggested exploring the use of Bacillus subtilis, a known probiotic often employed in aquaculture, as a potential chassis for our project.

 

Interview

Interviewee:

Prof. Zhangjie Chu

Institution:

School of Fishery, Zhejiang Ocean University

Field of research:

Aquatic product cultivation and water management

Suggestions and takeaways:

1) A. hydrophila spreads through water and tools, but proper disinfection and inspection help prevent outbreaks. 

2) Beneficial bacteria like B. subtilis can effectively inhibit pathogens, and strain optimization is key. 

3) Gene-modified B. subtilis can inhibit A. hydrophila quorum sensing, offering a sustainable alternative to antibiotics.

 

Following Prof. Chu's advice, we investigated B. subtilis and explored how to integrate it into our project. We consulted Prof. Jianmiao Xu, an expert in microbial metabolic engineering with extensive experience manipulating B. subtilis. He first confirmed that B. subtilis is an ideal chassis for producing quorum-quenching enzymes due to its non-pathogenic nature, ease of genetic manipulation, and high capacity for secretory protein production. He then suggested that we find an E. coli-B. subtilis shuttle secretion vector for exogenous gene expression. While working with B. subtilis in labs, we encountered issues with failed transformation using electroporation (see Engineering Success https://2024.igem.wiki/hangzhou-biox/engineering for more details). We sought further advice from Prof. Xu. He recommended trying chemical transformation using the Paris method. Following his suggestion, we successfully obtained transformants and were able to proceed.

 

Interview

Interviewee:

Prof. Jianmiao Xu

Institution:

College of Biotechnology and Bioengineering, Zhejiang University of Technology

Field of research:

Biocatalysis and microbial metabolic engineering

Suggestions and takeaways:

1) B. subtilis is an ideal chassis for quorum-quenching enzymes.

2) Use an E. coli-B. subtilis shuttle vector for efficient exogenous gene expression.

3) After failed electroporation attempts, Xu advised using the chemical transformation method (Paris method).

4) While enzyme production is well-established, Xu highlighted the challenge of optimizing enzyme concentration for aquaculture and balancing cost with environmental impact.

 

Finally, we interviewed Dr. Tianjiang Chu, an expert in indigenous fish preservation. He appreciated our focus on aquatic pathogens because they also pose a threat to wild indigenous fish. Dr. Chu suggested that our engineered B. subtilis could potentially help preserve indigenous aquatic species but emphasized the need for strict biosafety measures. He noted that genetically engineered organisms can impact ecosystems positively or negatively, so safety assessments are essential before use, and approval from authorities is required. This interview provided us with a new perspective on biodiversity, and we are glad that our project may contribute to this field in the future.

 

Interview

Interviewee:

Dr. Tianjiang Chu

Institution:

Aquatic Research Institute, Hangzhou Academy of Agricultural Sciences

Field of research:

Biodiversity and breeding of indigenous fish

Suggestions and takeaways:

1) Major threats to indigenous fish include invasive species, pollution, and pathogens.

2) Engineered B. subtilis should only be used in imbalanced ecosystems.

3) Dr. Chu’s team uses artificial breeding to conserve indigenous species, and our project may assist in combating pathogens in the artificial breeding process.