Difference between revisions of "Part:BBa K5208015"

 
 
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This is an expression cassette consisting of a strong inducible promoter Pgrac, a secretion signal peptide SPamyQ, and the AHL lactonase AhlS, and a terminator.
 
This is an expression cassette consisting of a strong inducible promoter Pgrac, a secretion signal peptide SPamyQ, and the AHL lactonase AhlS, and a terminator.
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'></span>
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===Sequence and Features===
 
<partinfo>BBa_K5208015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5208015 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K5208015 parameters</partinfo>
 
<partinfo>BBa_K5208015 parameters</partinfo>
 
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<!-- Add more about the biology of this part here-->
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===Usage and Biology===
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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).
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In this expression cassette, Pgrac([https://parts.igem.org/Part:BBa_K1628202 BBa_K1628202] is a strong promoter, and SPamyQ [https://parts.igem.org/Part:BBa_K1074014 BBa_K1074014] is a signal peptide that ensures the efficient secretion of the AHL lactonase. AhlS [https://parts.igem.org/Part:BBa_K5208005 BBa_K5208005] is an enzyme that catalyzes the hydrolysis of AHL. By expressing AhlS 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, [https://parts.igem.org/Part:BBa_K4934022 BBa_K4934022] is the terminator.
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===Characterization===
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'''2024 Hangzhou-BioX Team characterized this part for its quorum-quenching ability against A. hydrophila'''
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'''The zone of inhibition test'''
 +
 +
Our first test aimed to determine if B. subtilis WB600 expressing AhlS 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.
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<html>
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<body>
 +
<figure>
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<div class = "center">
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<center><img src = "https://static.igem.wiki/teams/5208/parts/15-1.jpg" style = "width:200px"></center>
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</div>
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<figcaption><center>Figure 1. The inhibition test of A. hydrophila was conducted with wells loaded with B. subtilis. LB served as the negative control. Chloramphenicol (Cm+) served as the positive control. </center></figcaption>
 +
</figure>
 +
</body>
 +
</html>
 +
 +
Results indicated that B. subtilis WB600 expressing AhlS 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.
 +
 +
<html>
 +
<body>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.wiki/teams/5208/parts/15-2.png" style = "width:500px"></center>
 +
</div>
 +
<figcaption><center>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. *: p < 0.05; **: p < 0.01; ***: p < 0.001. </center></figcaption>
 +
</figure>
 +
</body>
 +
</html>
 +
 +
Results showed that B. subtilis WB600 expressing AhlS significantly increased the AHL degradation ability (p < 0.001) (Figure 2B).
 +
 +
 +
 +
'''Natural AHL degradation test'''
 +
 +
Our experiment utilized the agar well diffusion method. Plates were prepared with molten LB agar mixed with CV026. Wells were loaded with a mixture of A. hydrophila and B. subtilis. CV026 in the agar detected the C4-HSL synthesized by A. hydrophila in the wells and responded by producing purple pigment, forming a purple ring around the wells. If B. subtilis secretes AHL lactonases, the C4-HSL synthesized by A. hydrophila would be degraded, resulting in a smaller purple area.
 +
 +
<html>
 +
<body>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.wiki/teams/5208/parts/15-3.png" style = "width:500px"></center>
 +
</div>
 +
<figcaption><center>Figure 3. A. Natural AHL degradation test on LB plates involved loading wells with a mixture of A. hydrophila and B. subtilis. Purple rings indicated the presence of C4-HSL; B. The AHL degradation levels of each strain were measured in the percentage reduction in ring size compared to the wells with A. hydrophila alone. *: p < 0.05; **: p < 0.01; ***: p < 0.001. </center></figcaption>
 +
</figure>
 +
</body>
 +
</html>
 +
 +
Results exhibited complete AHL degradation by AhlS (Figure 3).
 +
 +
 +
 +
'''Biofilm reduction test'''
 +
 +
The crystal violet assay was used to assess biofilm formation. To test the effect of B. subtilis expressing AhlS on A. hydrophila biofilm formation, A. hydrophila was incubated in partial B. subtilis culture supernatants under static conditions for 48 hours. The biofilm was then stained with crystal violet, and the dyed materials were homogenized to measure absorbance at 570 nm. To control for bacterial growth, we also measured OD600 immediately after the 48-hour incubation. The relative biofilm formation was normalized by calculating OD570/OD600 (Cam & Bicek, 2023; Parker et al., 2017).
 +
 +
<html>
 +
<body>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.wiki/teams/5208/parts/15-4.png" style = "width:500px"></center>
 +
</div>
 +
<figcaption><center>Figure 4. A. 48-hour A. hydrophila biofilm stained with crystal violet; B. Biofilm formation was quantified using OD570/OD600. *: p < 0.05; **: p < 0.01; ***: p < 0.001. </center></figcaption>
 +
</figure>
 +
</body>
 +
</html>
 +
 +
Results showed that B. subtilis WB600 expressing AhlS significantly reduced A. hydrophila biofilm formation by 78.8% (p < 0.001) (Figure 4B).
 +
 +
 +
 +
'''Extracellular protease reduction test'''
 +
 +
We measured the activity of the extracellular proteases of A. hydrophila cultured in B. subtilis AhlS supernatants using the Neutral Protease (NP) Activity Assay Kit (Sangon Biotech, Shanghai, China).
 +
 +
<html>
 +
<body>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.wiki/teams/5208/parts/15-5.png" style = "width:300px"></center>
 +
</div>
 +
<figcaption><center>Figure 5. The activity of the extracellular proteases of A. hydrophila cultured in partial B. subtilis supernatants. *: p < 0.05; **: p < 0.01; ***: p < 0.001.</center></figcaption>
 +
</figure>
 +
</body>
 +
</html>
 +
 +
The results demonstrated that AhlS significantly decreased the activity of A. hydrophila extracellular proteases (p < 0.05) (Figure 5).
 +
 +
 +
===References===
 +
Cam, S., & Bicek, S. (2023). The effects of temperature, salt, and phosphate on biofilm and exopolysaccharide production by Azotobacter spp. Arch Microbiol, 205(3), 87. https://doi.org/10.1007/s00203-023-03428-9
 +
 +
Coquant, G., Grill, J. P., & Seksik, P. (2020). Impact of N-Acyl-Homoserine Lactones, Quorum Sensing Molecules, on Gut Immunity. Front Immunol, 11, 1827. https://doi.org/10.3389/fimmu.2020.01827
 +
 +
Hlordzi, V., Kuebutornye, F. K. A., Afriyie, G., Abarike, E. D., Lu, Y., Chi, S., & Anokyewaa, M. A. (2020). The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquaculture Reports, 18, 100503. https://doi.org/https://doi.org/10.1016/j.aqrep.2020.100503
 +
 +
Khajanchi, B. K., Sha, J., Kozlova, E. V., Erova, T. E., Suarez, G., Sierra, J. C., Popov, V. L., Horneman, A. J., & Chopra, A. K. (2009). N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila. Microbiology (Reading), 155(Pt 11), 3518-3531. https://doi.org/10.1099/mic.0.031575-0
 +
 +
Parker, A., Cureoglu, S., De Lay, N., Majdalani, N., & Gottesman, S. (2017). Alternative pathways for Escherichia coli biofilm formation revealed by sRNA overproduction. Mol Microbiol, 105(2), 309-325. https://doi.org/10.1111/mmi.13702

Latest revision as of 07:04, 23 September 2024


Pgrac-SPamyQ-AhlS-Term

This is an expression cassette consisting of a strong inducible promoter Pgrac, a secretion signal peptide SPamyQ, and the AHL lactonase AhlS, 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. AhlS BBa_K5208005 is an enzyme that catalyzes the hydrolysis of AHL. By expressing AhlS 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 AhlS 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. Chloramphenicol (Cm+) served as the positive control.

Results indicated that B. subtilis WB600 expressing AhlS 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. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

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


Natural AHL degradation test

Our experiment utilized the agar well diffusion method. Plates were prepared with molten LB agar mixed with CV026. Wells were loaded with a mixture of A. hydrophila and B. subtilis. CV026 in the agar detected the C4-HSL synthesized by A. hydrophila in the wells and responded by producing purple pigment, forming a purple ring around the wells. If B. subtilis secretes AHL lactonases, the C4-HSL synthesized by A. hydrophila would be degraded, resulting in a smaller purple area.

Figure 3. A. Natural AHL degradation test on LB plates involved loading wells with a mixture of A. hydrophila and B. subtilis. Purple rings indicated the presence of C4-HSL; B. The AHL degradation levels of each strain were measured in the percentage reduction in ring size compared to the wells with A. hydrophila alone. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results exhibited complete AHL degradation by AhlS (Figure 3).


Biofilm reduction test

The crystal violet assay was used to assess biofilm formation. To test the effect of B. subtilis expressing AhlS on A. hydrophila biofilm formation, A. hydrophila was incubated in partial B. subtilis culture supernatants under static conditions for 48 hours. The biofilm was then stained with crystal violet, and the dyed materials were homogenized to measure absorbance at 570 nm. To control for bacterial growth, we also measured OD600 immediately after the 48-hour incubation. The relative biofilm formation was normalized by calculating OD570/OD600 (Cam & Bicek, 2023; Parker et al., 2017).

Figure 4. A. 48-hour A. hydrophila biofilm stained with crystal violet; B. Biofilm formation was quantified using OD570/OD600. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results showed that B. subtilis WB600 expressing AhlS significantly reduced A. hydrophila biofilm formation by 78.8% (p < 0.001) (Figure 4B).


Extracellular protease reduction test

We measured the activity of the extracellular proteases of A. hydrophila cultured in B. subtilis AhlS supernatants using the Neutral Protease (NP) Activity Assay Kit (Sangon Biotech, Shanghai, China).

Figure 5. The activity of the extracellular proteases of A. hydrophila cultured in partial B. subtilis supernatants. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

The results demonstrated that AhlS significantly decreased the activity of A. hydrophila extracellular proteases (p < 0.05) (Figure 5).


References

Cam, S., & Bicek, S. (2023). The effects of temperature, salt, and phosphate on biofilm and exopolysaccharide production by Azotobacter spp. Arch Microbiol, 205(3), 87. https://doi.org/10.1007/s00203-023-03428-9

Coquant, G., Grill, J. P., & Seksik, P. (2020). Impact of N-Acyl-Homoserine Lactones, Quorum Sensing Molecules, on Gut Immunity. Front Immunol, 11, 1827. https://doi.org/10.3389/fimmu.2020.01827

Hlordzi, V., Kuebutornye, F. K. A., Afriyie, G., Abarike, E. D., Lu, Y., Chi, S., & Anokyewaa, M. A. (2020). The use of Bacillus species in maintenance of water quality in aquaculture: A review. Aquaculture Reports, 18, 100503. https://doi.org/https://doi.org/10.1016/j.aqrep.2020.100503

Khajanchi, B. K., Sha, J., Kozlova, E. V., Erova, T. E., Suarez, G., Sierra, J. C., Popov, V. L., Horneman, A. J., & Chopra, A. K. (2009). N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila. Microbiology (Reading), 155(Pt 11), 3518-3531. https://doi.org/10.1099/mic.0.031575-0

Parker, A., Cureoglu, S., De Lay, N., Majdalani, N., & Gottesman, S. (2017). Alternative pathways for Escherichia coli biofilm formation revealed by sRNA overproduction. Mol Microbiol, 105(2), 309-325. https://doi.org/10.1111/mmi.13702