Part:BBa_K5208010

Pgrac-SPamyQ-AiiA-Term

This is an expression cassette consisting of a strong inducible promoter Pgrac, a secretion signal peptide SPamyQ, the AHL lactonase AiiA, and a terminator.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Aeromonas hydrophila is an aquatic pathogen that poses a significant threat to the aquaculture industry. Its virulence is primarily regulated by quorum sensing, a mechanism triggered by acyl-homoserine lactones (AHLs), particularly N-butanoyl-L-homoserine lactone (C4-HSL) (Coquant et al., 2020; Hlordzi et al., 2020).

In this expression cassette, Pgrac BBa_K1628202 is a strong promoter, and SPamyQ BBa_K1074014 is a signal peptide that ensures the efficient secretion of the AHL lactonase. AiiA BBa_K5208000 is an enzyme that catalyzes the hydrolysis of AHL. By expressing AiiA in the probiotic Bacillus subtilis and introducing the engineered bacteria into fish ponds, quorum sensing in A. hydrophila can be disrupted, reducing its virulence and safeguarding aquatic animals. Finally, BBa_K4934022 is the terminator.

Characterization

2024 Hangzhou-BioX Team characterized this part for its quorum-quenching ability against A. hydrophila The zone of inhibition test Our first test aimed to determine if B. subtilis WB600 expressing AiiA directly inhibits the growth of A. hydrophila. Our experiment utilized the agar well diffusion method. We prepared LB agar plates containing 10% overnight culture of A. hydrophila and loaded B. subtilis cultures into the wells.

Figure 1. The inhibition test of A. hydrophila was conducted with wells loaded with B. subtilis. LB served as the negative control.

Results indicated that B. subtilis WB600 expressing AiiA did not inhibit the growth of A. hydrophila (Figure 1), since quorum quenching does not kill bacteria but only influences the expression of certain virulence factors (Khajanchi et al., 2009).

Synthetic AHL degradation test Wells were punched into LB agar plates containing synthetic C4-HSL and Chromobacterium subtsugae CV026 (the biosensor for AHL, turns purple when detecting AHL). IPTG-induced B. subtilis cultures were loaded into the wells. The AHL lactonases secreted by the bacteria diffused into the agar, degrading the C4-HSL. Consequently, areas around the wells lacked the characteristic purple color.

Figure 2. A. Synthetic AHL degradation test on LB plates. B. subtilis cultures were loaded in wells. Liquid LB was used as the negative control. Rings without the purple color indicated AHL degradation; B. AHL degradation levels of each strain were measured in the width of the colorless ring; C. AHL degradation levels of mixed sample groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results showed that B. subtilis WB600 expressing AiiA significantly increased the AHL degradation ability (p < 0.001) (Figure 2B).

For the mixed tests combining AiiA with other AHL lactonases (YtnP and AiiM), all data points for the mixed groups fell between the values of the individual strains (Figure 2C). This suggests that no enzyme-enzyme interactions occurred among the AHL lactonases tested.