Difference between revisions of "Part:BBa K2599001"
 
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Figure 1 biobrick picture
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{{#tag:html|<img style="width: 20%; padding-left: 40%;" src="https://static.igem.org/mediawiki/2018/f/f2/T--NCTU_Formosa--Bov.png" alt="" />}}
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<div style="width:40%; padding-left: 30%;"><p style="padding-top: 10px; font-size: 10px; text-align: center;"><b>Figure 1.</b> Basic part of Bovicin HJ50</p></div>
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<p style="padding-top:20px;font-size:20px"><b>Introduction</b></p>
 
<p style="padding-top:20px;font-size:20px"><b>Introduction</b></p>
  
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Bovicin HJ50 is isolated from <i> Streptococcus bovis </i> HJ50. It contains a disulfide bridge and shows similarity to type AII lantibiotics, the largest group of lantibiotics. Like most of the bacteriocins produced by lactic acid bacteria, Bovicin HJ50 showed a narrow range of inhibiting activity. Its antimicrobial activity has been proved in reference.
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Bovicin HJ50 is isolated from <i> Streptococcus bovis </i> HJ50. It shows similarity to type IIa lantibiotics, the largest group of lantibiotics. It comprises a linear N-terminal region and a globular C-terminal region. Its disulfide bond is especially essential for antimicrobial activity. Like most of the bacteriocins produced by lactic acid bacteria, Bovicin HJ50 showed a narrow range of inhibiting activity. Its antimicrobial activity has been proved in reference.
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 +
 
 +
 
 +
<p style="padding-top:20px;font-size:20px"><b>Mechanism of Bovicin HJ50</b></p>
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The bacteriocins inhibit their target organisms through pore formation. Though the mechanism of each inhibition is vary from species to species, the general process is conserved. To see more details, please search for our project page.
 +
 
 +
The bactericidal activity of Bovicin HJ50 is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores. Its pore-forming activity is significantly different from other lantibiotics, suggesting a novel antimicrobial mechanism.
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 +
 
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{{#tag:html|<img style="width: 25%; padding-left: 38%;" src="https://static.igem.org/mediawiki/2018/b/bc/T--NCTU_Formosa--mechanism.png" alt="" />}}
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<div style="width:40%; padding-left: 30%;"><p style="padding-top: 20px; font-size: 10px; text-align: center;"><b>Figure 2.</b> Mechanism of bacteriocin</p></div>
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<p style="padding-top:10px;font-size:20px;"><b>Features of Bovicin HJ50</b></p>
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<p style="padding-top:16px;font-size:16px"><b>1. Species Specific</b></p>
 +
 
 +
Bacteriocins target strains or closely related species. The organisims that Bovicin HJ50 targets including <i>Bacillus megaterium</i>, <i>Bacillus subtilis</i>, <i>Bacillus coagulans</i>, etc. Thus this bacteriocin is one of the peptide candidates for our project, that can solve the unbalance microbiota of agriculture in Taiwan.
 +
 
 +
More target organisms can be found on [http://bactibase.hammamilab.org/BAC156 bactibase].
 +
 
 +
<p style="padding-top:16px;font-size:16px"><b>2. Eco-friendly</b></p>
 +
 
 +
Since Bovicin HJ50 is a polypeptide naturally produced by bacteria itself and can inhibit other bacteria without much environment impact. It don't pose threat to other organisms like farm animals or humans. Therefore, this toxin will not cause safety problem.
 +
 
 +
<p style="padding-top:16px;font-size:16px"><b>3. Biodegradable</b></p>
 +
 
 +
Bovicin HJ50 is a short peptide that will degrade in a short time. After degradation, this antibacterial peptide is harmless to our environment.
 +
 
 +
 
 +
<p style="padding-top:10px;font-size:20px;"><b>Peptide Prediction</b></p>
 +
 
 +
NCTU_Formosa 2017 had compeleted a [http://2017.igem.org/Team:NCTU_Formosa/Model peptide prediction model] that can predict
 +
peptide for new function. In this model, they used scoring card method (SCM) for machine learning. This year, NCTU_Formosa 2018 continued to use the same method for predicting antimicrobial peptide, in order to seek more candidates for our project.
 +
 
 +
Bovicin HJ50 is one of the existing peptides that we predicted to show the function of antimicrobial activity. The score of our prediction is 459.87.
 +
 
 +
 
 +
{{#tag:html|<img style="width: 70%; padding-left: 15%;" src="" alt="" />}}
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<div style="width:40%; padding-left: 30%;"><p style="padding-top: 10px; font-size: 10px; text-align: center;"><b>Figure 3.</b> The prediction result of Bovicin HJ50.</p></div>
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===Cloning===
 
===Cloning===
  
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We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The Bovicin HJ50 sequence length is around 171 b.p.
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We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The Bovicin HJ50 sequence length is around 171 b.p. and the length of PCR product should be around 221 b.p.
  
  
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Figure 2 PCR
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{{#tag:html|<img style="width: 20%; padding-left: 40%;" src="https://static.igem.org/mediawiki/2018/0/00/T--NCTU_Formosa--Bov_basic.png" alt="" />}}
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<div style="width:40%; padding-left: 30%;"><p style="padding-top: 10px; font-size: 10px; text-align: center;"><b>Figure 4.</b> Agarose gel electrophoretic pattern of Taq PCR product.</p></div>
  
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More experiment results of Bovicin HJ50, please look for the composite part [https://parts.igem.org/Part:BBa_K2599009 BBa_K2599009].
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<p style="padding-top:10px;font-size:20px;"><b>Safety</b></p>
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In the future, we are going to spray our bio-stimulator into the environment. To make sure whether the bacteria contain anti-microbial peptide will not exist in the final product, we design the processing standards in the laboratory.
 +
 +
Bacteriocins are usually heat stable, we use high-temperature sterilization to double make sure our peptide solution does not contain any living E. coli. However, peptides may degrades after long time sterilization. To find out the best fitted time for sterilization, we boiled our bacteriocins for 0, 15, 30, and 45 minutes, and put them on LB Agar plate and cultured it at 37℃ for 16 hours.
 +
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From the result of the plate, we can easily observe that bacteria exists only in the sample that is not boiled. After fifteen minutes of sterilization, there are no alive bacterias exist.
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{{#tag:html|<img style="width: 30%; padding-left: 35%;" src="https://static.igem.org/mediawiki/2018/a/a2/T--NCTU_Formosa--Bov_safety_plate.png" alt="" />}}
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<div style="width:70%; padding-left: 15%;"><p style="padding-top: 10px; font-size: 10px; text-align: center;"><b>Figure 5.</b> LB Agar plate of sterilization of Bovicin HJ50+intein+CBD. (A)Negative control:LB broth. (B)Sterilize for 0 minutes. (C)Sterilize for 15 minutes. (D)Sterilize for 30 minutes. (E)Sterilize for 45 minutes.  </p></div>
  
  
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<partinfo>BBa_K2599001 parameters</partinfo>
 
<partinfo>BBa_K2599001 parameters</partinfo>
 
<!-- -->
 
<!-- -->
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<p style="padding-top:20px;font-size:20px"><b>Reference</b></p>
 
<p style="padding-top:20px;font-size:20px"><b>Reference</b></p>
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1. Liu, G., et al. (2009). "Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50." Microbiology 155(Pt 2): 584-593.
 +
 +
2. Xiao, H., et al. (2004). "Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50." Microbiology 150(Pt 1): 103-108.
 +
 +
3. Zhang, J., et al. (2014). "Type AII lantibiotic bovicin HJ50 with a rare disulfide bond: structure, structure-activity relationships and mode of action." Biochem J 461(3): 497-508.

Latest revision as of 18:59, 17 October 2018


Antimicrobial peptide - Bovicin HJ50

This biobrick is the basic part of the Bovicin HJ50, more information please look for the composite part (BBa_K2599009).



Figure 1. Basic part of Bovicin HJ50


Introduction

Bovicin HJ50 is isolated from Streptococcus bovis HJ50. It shows similarity to type IIa lantibiotics, the largest group of lantibiotics. It comprises a linear N-terminal region and a globular C-terminal region. Its disulfide bond is especially essential for antimicrobial activity. Like most of the bacteriocins produced by lactic acid bacteria, Bovicin HJ50 showed a narrow range of inhibiting activity. Its antimicrobial activity has been proved in reference.


Mechanism of Bovicin HJ50

The bacteriocins inhibit their target organisms through pore formation. Though the mechanism of each inhibition is vary from species to species, the general process is conserved. To see more details, please search for our project page.

The bactericidal activity of Bovicin HJ50 is based on depolarization of energized bacterial cytoplasmic membranes, initiated by the formation of aqueous transmembrane pores. Its pore-forming activity is significantly different from other lantibiotics, suggesting a novel antimicrobial mechanism.


Figure 2. Mechanism of bacteriocin


Features of Bovicin HJ50

1. Species Specific

Bacteriocins target strains or closely related species. The organisims that Bovicin HJ50 targets including Bacillus megaterium, Bacillus subtilis, Bacillus coagulans, etc. Thus this bacteriocin is one of the peptide candidates for our project, that can solve the unbalance microbiota of agriculture in Taiwan.

More target organisms can be found on [http://bactibase.hammamilab.org/BAC156 bactibase].

2. Eco-friendly

Since Bovicin HJ50 is a polypeptide naturally produced by bacteria itself and can inhibit other bacteria without much environment impact. It don't pose threat to other organisms like farm animals or humans. Therefore, this toxin will not cause safety problem.

3. Biodegradable

Bovicin HJ50 is a short peptide that will degrade in a short time. After degradation, this antibacterial peptide is harmless to our environment.


Peptide Prediction

NCTU_Formosa 2017 had compeleted a [http://2017.igem.org/Team:NCTU_Formosa/Model peptide prediction model] that can predict peptide for new function. In this model, they used scoring card method (SCM) for machine learning. This year, NCTU_Formosa 2018 continued to use the same method for predicting antimicrobial peptide, in order to seek more candidates for our project.

Bovicin HJ50 is one of the existing peptides that we predicted to show the function of antimicrobial activity. The score of our prediction is 459.87.


Figure 3. The prediction result of Bovicin HJ50.


Experiment Result

Cloning

We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The Bovicin HJ50 sequence length is around 171 b.p. and the length of PCR product should be around 221 b.p.


Figure 4. Agarose gel electrophoretic pattern of Taq PCR product.


More experiment results of Bovicin HJ50, please look for the composite part BBa_K2599009.


Safety

In the future, we are going to spray our bio-stimulator into the environment. To make sure whether the bacteria contain anti-microbial peptide will not exist in the final product, we design the processing standards in the laboratory.

Bacteriocins are usually heat stable, we use high-temperature sterilization to double make sure our peptide solution does not contain any living E. coli. However, peptides may degrades after long time sterilization. To find out the best fitted time for sterilization, we boiled our bacteriocins for 0, 15, 30, and 45 minutes, and put them on LB Agar plate and cultured it at 37℃ for 16 hours.

From the result of the plate, we can easily observe that bacteria exists only in the sample that is not boiled. After fifteen minutes of sterilization, there are no alive bacterias exist.


Figure 5. LB Agar plate of sterilization of Bovicin HJ50+intein+CBD. (A)Negative control:LB broth. (B)Sterilize for 0 minutes. (C)Sterilize for 15 minutes. (D)Sterilize for 30 minutes. (E)Sterilize for 45 minutes.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 20
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

1. Liu, G., et al. (2009). "Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50." Microbiology 155(Pt 2): 584-593.

2. Xiao, H., et al. (2004). "Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50." Microbiology 150(Pt 1): 103-108.

3. Zhang, J., et al. (2014). "Type AII lantibiotic bovicin HJ50 with a rare disulfide bond: structure, structure-activity relationships and mode of action." Biochem J 461(3): 497-508.