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==Contribution: iGEM22_WHU-China ==
 
==Contribution: iGEM22_WHU-China ==
  
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WHU-China 2022 further investigated about the properties of PBSX holin. PBSX holin ([[Part:BBa_K1054002]]) is a small membrane protein in double-stranded DNA phages for efficient host lysis. Holins are the gatekeepers of the lysis process, possessing an intriguing ability to be triggered, at a precise time point, to form large holes in the cytoplasmic membrane of phage-infected bacteria. [1] <br>
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PBSX holin is a small membrane protein in double-stranded DNA phages for efficient host lysis. Previous research has found that Holins are the gatekeepers of the lysis process, possessing an intriguing ability to be triggered, at a precise time point, to form large holes in the cytoplasmic membrane of phage-infected bacteria. ([1] Mart Krupovič, Dennis H. Bamford. 2008) <br>
  
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Because of the properties of holin mentioned above, we think that holin is effective and secure for our kill switch. It cause the cleavage of the engineered bacteria only, releasing no toxin in the gut or environment.<br>
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                <p style="color:Gray; padding:0px 30px 10px;">Figure. 1 The ‘death raft’ model for holin lesion formation. (Mart Krupovič, Dennis H. Bamford. 2008)</p>
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In this research, a ‘death raft’ model is used to describe holin lesion formation. The process includes accumulation and oligomerization of holins in the CM, formation of the ‘death raft’, the opening of an aqueous channel and formation of the holin lesion. (Figure. 1)
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After understanding the functional mechanism of holin, we predicted holin’s structure by PolyPhobius prediction. It shows that PBSX holin has two transmembrane regions. (Figure. 2) <br>
  
 
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                 <p style="color:Gray; padding:0px 30px 10px;">Figure 1:</b> A.The prediction of transmembrane domain of PBSX holin.
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                 <p style="color:Gray; padding:0px 30px 10px;">Figure. 2 The prediction of the transmembrane domain of PBSX holin.</p>
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B.The ‘death raft’ model for holin lesion formation. (Mart Krupovič, Dennis H. Bamford. 2008)</p>
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The structure is predicted by PolyPhobius prediction. It show that PBSX holin has two transmemebrane region. (fig 1.A)
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Inspired by the properties of holin mentioned above, we think holin is an effective and secure choice for our kill switch, for it will cause the cleavage of the engineered bacteria only, releasing no toxin in the gut or environment.<br>
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A ‘death raft’ model is used to describe holin lesion formation. Th process includes accumulation and oligomerization of holins in the CM, formation of the ‘death raft’, opening of an aqueous channel and formation of the holin lesion. (fig 1.B) <br>
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We induced the expression of PBSX holin in E. coli at the log phase and measured OD600. The result shows the ability of holin to inhibit the growth of bacteria to some extent. (Figure. 3)
  
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We induced the expression of PBSX holin in E.coli at the log phase and measured OD. The result shows the ability of holin to inhibit the growth of bacteria to some extent.(fig. 2)<br>
 
  
 
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                 <p style="color:Gray; padding:0px 30px 10px;">Figure. 2: Inducing the expression of PBSX holin in E. coli. A. Induction on the culture plate. B. Change in turbidity.</p>
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                 <p style="color:Gray; padding:0px 30px 10px;">Figure. 3: Induction of the expression of PBSX holin in E. coli. A.Induction on a culture plate. B. Change in turbidity.</p>
 
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Reference:<br>
 
Reference:<br>

Latest revision as of 13:37, 13 October 2022

bacteriophage PBSX holin

Holin is an essential protein for host lysis by bacteriophage. This part is cloned from Bacillus subtilis subsp. subtilis str. 168. Its activity can by blocked by another protein called lrgA (BBa_K1054024) and lrgB (BBa_K1054003). We can use this feature to prevent horizontal gene transfer of certain plasmid.

Holin 2013zjuchina.png

Growth curve of BL21 E.coli cells induced by different concentrations of IPTG

Contribution: iGEM22_WHU-China

PBSX holin is a small membrane protein in double-stranded DNA phages for efficient host lysis. Previous research has found that Holins are the gatekeepers of the lysis process, possessing an intriguing ability to be triggered, at a precise time point, to form large holes in the cytoplasmic membrane of phage-infected bacteria. ([1] Mart Krupovič, Dennis H. Bamford. 2008)

Figure. 1 The ‘death raft’ model for holin lesion formation. (Mart Krupovič, Dennis H. Bamford. 2008)

In this research, a ‘death raft’ model is used to describe holin lesion formation. The process includes accumulation and oligomerization of holins in the CM, formation of the ‘death raft’, the opening of an aqueous channel and formation of the holin lesion. (Figure. 1)

After understanding the functional mechanism of holin, we predicted holin’s structure by PolyPhobius prediction. It shows that PBSX holin has two transmembrane regions. (Figure. 2)

Figure. 2 The prediction of the transmembrane domain of PBSX holin.


Inspired by the properties of holin mentioned above, we think holin is an effective and secure choice for our kill switch, for it will cause the cleavage of the engineered bacteria only, releasing no toxin in the gut or environment.

We induced the expression of PBSX holin in E. coli at the log phase and measured OD600. The result shows the ability of holin to inhibit the growth of bacteria to some extent. (Figure. 3)


Figure. 3: Induction of the expression of PBSX holin in E. coli. A.Induction on a culture plate. B. Change in turbidity.


Reference:
[1] Mart Krupovicˇ and Dennis H. Bamford. Holin of bacteriophage lambda: structural insights into a membrane lesion. Molecular Microbiology(2008)69(4), 781–783.

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]