iGEM 2024 — Team hangzhou-biox


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### About Our Synth Bio

# Engineering

## **A****bstract**

Our project aimed to reduce the virulence of *Aeromonas hydrophila*, a harmful aquatic pathogen, to safeguard the aquaculture
industry. This pathogen uses AHL molecules to activate quorum
sensing, leading to the expression of virulence factors. We
introduced six AHL lactonases from various bacteria donors into *Bacillus subtilis*. By degrading AHL, our engineered *B. subtilis* effectively inhibits the quorum sensing of *A.* *hydrophila*, significantly reducing both biofilm formation and extracellular
protease activity, with the strain expressing AiiA showing the
most pronounced effect. This part is structured in three DBTL cycles.

## **D****BTL Cycle 1**

**2****. Build**

**3.** **Test**

As we followed the electroporation protocols, colony growth on
selective plates was observed, but no positive transformants
were detected in colony PCR tests. We repeated the
transformation several times with tuned voltage, plasmid amount,
and buffer concentrations. Yet, results remained consistently
unsuccessful.

**4.** **Learn**

Interestingly, *B. subtilis* appeared to develop antibiotic resistance (we tried
kanamycin and chloramphenicol) even in negative controls without
plasmid introduction. Literature suggests that cellular stress
can induce antibiotic tolerance in bacteria (Nguyen et al., 2011; Poole, 2012; Yang et al., 2006). We hypothesize that the electric shock-induced stress
responses in *B. subtilis*, allowing growth on selective plates and leading to false
positive results.

Given the unresolved issues with electroporation and time
constraints, we decided to switch to chemical
transformation.

## **D****BTL Cycle 2**

**3****. Test**

## **DBTL Cycle 3**

**2****. Build**

The engineered *B. subtilis* strains expressing exogenous AHL lactonases have been
successfully constructed in the previous cycle. The setups for
AHL and quorum-related tests were prepared as described in the
Design.

**3. Test**

**4****. Learn**

We proved that even the original *B. subtilis* WB600 strain exhibited some ability to degrade C4-HSL. We
learned that by introducing BsAiiA, BsYtnP, MtAiiM, AsAhlD, and
SsAhlS, the C4-HSL degrading ability of WB600 increased
dramatically, whereas AtAttM does not degrade C4-HSL
efficiently. Additionally, we showed that BsAiiA, BsYtnP, and
MtAiiM functioned independently without enzyme-enzyme
interactions, and mixing two enzymes did not yield superior
results compared to the individual enzymes. We also confirmed
that quorum quenching does not inhibit the growth of *A. hydrophila*, but it does significantly reduce biofilm formation and
extracellular protease activity. In conclusion, we found that *B. subtilis* WB600 expressing AiiA is the most effective quorum
quenching strain against *A. hydrophila*, making it a promising probiotic for aquaculture
applications.

Due to iGEM’s policy discouraging animal experiments on
vertebrates, we chose not to collect direct mortality data on
aquatic animals. Instead, we found studies employing similar
strategies to control *A. hydrophila* have conducted animal experiments, so we performed a
survival analysis using their mortality data (Chen et al., 2020; Chu et al., 2014). Specifically, we referenced a study that used a
quorum-quenching *Bacillus* strain (*Bacillus sp.* QSI-1) to observe death rate changes in *A. hydrophila*-infected zebrafish (Figure 14).

![](https://static.igem.wiki/teams/5208/engineering/0926/16.png)

Figure 14. Survival rates of *A. hydrophila-*infected zebrafish with or without *Bacillus sp.* QSI-1 (Chu et al., 2014).

The results showed a significant difference in zebrafish
survival rates between the two groups (p = 0.02), suggesting
that the quorum-quenching strain effectively protects against *A. hydrophila* infection. This result further validates that developing
our engineered *B. subtilis* is a feasible approach for protecting aquatic animals
from *A. hydrophila*.

**5****. Future plans**

Our first plan focuses on biosafety measures for engineered *B. subtilis*. Although both the bacterium and the introduced enzymes are
generally safe, preventing environmental leakage is still
critical. We aim to introduce a suicide switch that activates
under dry and low-temperature conditions, ensuring that the
engineered bacteria can survive only in aquaculture ponds, which
are maintained at warm temperatures even in winter. The bacteria
will die when exposed to non-aquatic environments due to dry
conditions or natural water bodies during winter due to low
temperatures.

To achieve this, we plan to use the TreA promoter and the Des
promoter from *B. subtilis*. The TreA promoter is activated by osmotic stress and
dehydration (Sniezko et al., 1998; Xiao et al., 2024), and the Des promoter responds to low temperatures (Cybulski et al., 2004; Thuy Le & Schumann, 2007). We will place the MazF-MazE toxin-antitoxin suicide system
under the control of these promoters. This well-established
toxin-antitoxin system has been validated by several iGEM teams,
including 2010\_ Newcastle, 2023\_Freiburg, 2023\_ GEMS-Taiwan, etc. This design should ensure that the bacteria
die under temperature and osmotic stress, effectively limiting
their survival outside of controlled aquaculture
environments.

Second, we plan to measure the influence of our engineered *B. subtilis* on other virulence factors of *A. hydrophila*, including aerolysin and hemolysin, which are two critical
toxins involved in the bacterium’s pathogenicity (Khajanchi et al., 2009; Swift et al., 1997). This year, we were unable to acquire the red blood cells
necessary for testing these two toxins.

Finally, we plan to test the engineered *B. subtilis* against other aquatic bacterial pathogens, including *Edwardsiella tarda* and *Vibrio alginolyticus* (Irshath et al., 2023), as well as any other pathogens that utilize quorum sensing
for virulence factor expression. By doing so, we can explore
broader applications for our bacteria in real-world
settings.

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