Difference between revisions of "Part:BBa K2599008"
 
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<partinfo>BBa_K2599008 short</partinfo>
 
<partinfo>BBa_K2599008 short</partinfo>
  
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NCTU_Formosa 2018 designed a composite part encoding the Subtilosin sequence [https://parts.igem.org/Part:BBa_K2599000 (BBa_K2599000)], and then combined with a T7 promoter [https://parts.igem.org/Part:BBa_I712074 (BBa_I712074)], a ribosome binding site [https://parts.igem.org/Part:BBa_B0034 (BBa_B0034)], intein and chintin binding domain (CBD). Further information of our peptide can be found on our design page.
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NCTU_Formosa 2018 designed a composite part encoding the Subtilosin sequence [https://parts.igem.org/Part:BBa_K2599000 (BBa_K2599000)], and then combined with a T7 promoter [https://parts.igem.org/Part:BBa_I712074 (BBa_I712074)], a lac operator [https://parts.igem.org/Part:BBa_K1624002 (K1624002)], a ribosome binding site [https://parts.igem.org/Part:BBa_B0034 (BBa_B0034)], intein and chintin binding domain (CBD) [https://parts.igem.org/Part:BBa_K1465230 (BBa_K1465230)]. Further information of our peptide can be found on our design page.
  
  
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Figure 1 biobrick picture
 
  
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<h1>'''Introduction:'''</h1>
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{{#tag:html|<img style="width: 60%; padding-left: 18%;" src="https://static.igem.org/mediawiki/2018/b/bf/T--NCTU_Formosa--Sub_2.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> Composite part of Subtilosin</p></div>
  
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<i> Bacillus subtilis </i> produces a bacteriocin called Subtilosin that possesses antibacterial activity against certain gram-positive bacteria. The bacteriocins are a group of anitmicrobial peptides that are often distinguished from traditioinal antibiotics by their narrow range of avtivity against closely related bacteria.
 
  
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<p style="padding-top:20px;font-size:20px"><b>Introduction</b></p>
  
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===Mechanism of Subtilosin===
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<i> Bacillus subtilis </i> produces a bacteriocin called Subtilosin that targets the surface receptors and electrostatically binds to the bacterial cell membrane. Bacteriocins are a group of anitmicrobial peptides that are often distinguished from traditioinal antibiotics by their narrow range of avtivity against closely related bacteria.
  
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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. The details is on our design page.
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Subtilosin was evaluated as a quorum sensing (QS) inhibitor in gram-positive bacteria using Fe(III) reduction assay. In gram-positive bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter.
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<p style="padding-top:20px;font-size:20px"><b>Mechanism of Subtilosin</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. Details are on our project page.
  
 
According to the reference, Subtilosin acts by fully depleting the transmembrane pH gradient (ΔpH) and causing an immediate efflux of intracellular ATP, but has no effect on the transmembrane electric potential. In addition, the paper also shows that membrane permeabilization occurred at concentrations of subtilosin that were significantly higher than the MIC level of <i>E. coli</i> tested strains.
 
According to the reference, Subtilosin acts by fully depleting the transmembrane pH gradient (ΔpH) and causing an immediate efflux of intracellular ATP, but has no effect on the transmembrane electric potential. In addition, the paper also shows that membrane permeabilization occurred at concentrations of subtilosin that were significantly higher than the MIC level of <i>E. coli</i> tested strains.
  
  
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===Experiment Result===
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<p style="padding-top:10px;font-size:20px;"><b>Features of Subtilosin</b></p>
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<p style="padding-top:16px;font-size:16px"><b>1. Species Specific</b></p>
  
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<b>Cloning</b>
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Bacteriocin target strains or closely related species. Subtilosin has specificity to <i> Bacillus subtilis </i>, <i> Listeria monocytogenes </i>, <i> Enterococcus faecalis </i>, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC098 bactibase].
  
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We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The Subtilosin sequence length is around 147 b.p. For the composite part, the sequence length should be near at 1191 b.p.
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<p style="padding-top:16px;font-size:16px"><b>2. Eco-friendly</b></p>
  
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Since subtilosin 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.
  
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Figure 2 PCR
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<p style="padding-top:16px;font-size:16px"><b>3. Biodegradable</b></p>
  
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Subtilosin is a short peptide that will degrade in a short time. After degradation, this antibacterial peptide is harmless to our environment.
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<p style="padding-top:10px;font-size:20px;"><b>Experiment Result</b></p>
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===Cloning===
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We conbined our toxic gene to pSB1C3 backbone by the two restriction sites, EcoRI and SpeI, and conducted PCR to check the size of our part. The Subtilosin sequence length is around 147 b.p. For the composite part, the sequence length should be near at 1191 b.p. There are also some restrictioin sites at the two sides of our target protein, provided for future team to utilize the intein tag.
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{{#tag:html|<img style="width: 20%; padding-left: 40%;" src="https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Sub_comp.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 2.</b> PCR product </p></div>
  
  
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<b>Expressing</b>
 
  
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We chose <i> E. coli </i> 2566 strain to express our antibacterial peptides. The expression of Subtilosin fused with intein was induced by IPTG in <i> E. coli </i>, and intein-subtilosin specifically bound to the column through chitin binding domain would be purified.
 
  
  
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Figure 3 SDS
 
  
  
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<partinfo>BBa_K2599008 parameters</partinfo>
 
<partinfo>BBa_K2599008 parameters</partinfo>
 
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<p style="padding-top:20px;font-size:20px"><b>Reference</b></p>

Latest revision as of 01:54, 2 October 2018


T7 Promoter+RBS+Subtilosin+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Subtilosin sequence (BBa_K2599000), and then combined with a T7 promoter (BBa_I712074), a lac operator (K1624002), a ribosome binding site (BBa_B0034), intein and chintin binding domain (CBD) (BBa_K1465230). Further information of our peptide can be found on our design page.



Figure 1. Composite part of Subtilosin


Introduction

Bacillus subtilis produces a bacteriocin called Subtilosin that targets the surface receptors and electrostatically binds to the bacterial cell membrane. Bacteriocins are a group of anitmicrobial peptides that are often distinguished from traditioinal antibiotics by their narrow range of avtivity against closely related bacteria.

Subtilosin was evaluated as a quorum sensing (QS) inhibitor in gram-positive bacteria using Fe(III) reduction assay. In gram-positive bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter.


Mechanism of Subtilosin

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. Details are on our project page.

According to the reference, Subtilosin acts by fully depleting the transmembrane pH gradient (ΔpH) and causing an immediate efflux of intracellular ATP, but has no effect on the transmembrane electric potential. In addition, the paper also shows that membrane permeabilization occurred at concentrations of subtilosin that were significantly higher than the MIC level of E. coli tested strains.


Features of Subtilosin

1. Species Specific

Bacteriocin target strains or closely related species. Subtilosin has specificity to Bacillus subtilis , Listeria monocytogenes , Enterococcus faecalis , etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC098 bactibase].

2. Eco-friendly

Since subtilosin 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

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


Experiment Result

Cloning

We conbined our toxic gene to pSB1C3 backbone by the two restriction sites, EcoRI and SpeI, and conducted PCR to check the size of our part. The Subtilosin sequence length is around 147 b.p. For the composite part, the sequence length should be near at 1191 b.p. There are also some restrictioin sites at the two sides of our target protein, provided for future team to utilize the intein tag.


Figure 2. PCR product





Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1055
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 778
    Illegal AgeI site found at 868
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 698



Reference