Difference between revisions of "Part:BBa K3552005"

 
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__NOTOC__
  
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<partinfo>BBa_K3552005 short</partinfo>
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Prepilin peptidase-dependent protein A, a minor pilin. A bitopic inner membrane protein located in the inner membrane, with the length of 471 bp/156 aa. prepilin peptidase-dependent protein B, a minor pilin. a bitopic inner membrane protein located in the cytosol of the inner membrane, with the length of 564 bp/ 187 aa. YgdB is a domain-containing protein with a length of 408 bp/135 aa. prepilin peptidase-dependent protein C, a bitopic inner membrane protein located in the inner membrane, with the length of 324 bp/ 107 aa. GspO plays a role in type II pseudopili formation by removing the leader sequence from substrate proteins and subsequently methylating the alpha-amino group of the newly exposed N-terminal phenylalanine. Substrates include proteins required for biogenesis of the type II general secretory apparatus. This part is in the part collection where we have 13 genes that code for the proteins.
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The part collection includes:
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Parts that are different kinds of type 4 pilus:
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<partinfo>BBa_K3552003</partinfo> 
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<partinfo>BBa_K3552004</partinfo>
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<partinfo>BBa_K3552005</partinfo>
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<partinfo>BBa_K3552006</partinfo>
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<partinfo>BBa_K3552007</partinfo>
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<partinfo>BBa_K3552008</partinfo>
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<partinfo>BBa_K3552018</partinfo> 
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<partinfo>BBa_K3552019</partinfo> 
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<partinfo>BBa_K3552020</partinfo> 
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<partinfo>BBa_K3552021</partinfo> 
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<partinfo>BBa_K3552022</partinfo> 
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<partinfo>BBa_K3552023</partinfo> 
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<partinfo>BBa_K3552024</partinfo> 
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<partinfo>BBa_K3552025</partinfo> 
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<partinfo>BBa_K3552026</partinfo> 
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<partinfo>BBa_K3552027</partinfo> 
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<partinfo>BBa_K3552028</partinfo> 
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<partinfo>BBa_K3552029</partinfo> 
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Our part collection can instruct other teams to designed new rechargeable pilus and substitution of different major pilin.
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<span class='h3bb'>'''Sequence and Features'''</span>
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<partinfo>BBa_K3552005 SequenceAndFeatures</partinfo>
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==Usage and Biology==
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The ppdAB-ygdB-ppdC operon encodes the four minor pilins. These minor pilins and their homologues in the family contain two conserved Cys pairs at similar positions. We used the ppd genes from the EHEC strain EDL933 as building blocks to reconstitute an artificial T4P assembly operon in non-pathogenic E.coli, DH5a. Neither deletion of ppdC nor overproduction of ppdABC results in defects in chemotais or flagellar motility. This gene is predicted to be an inner membrane protein. The gene gspO, one of the two prepilin peptidase genes in E.coli K-12, is the last gene of the gsp operon encoding the chitinase-specific type 2 secretion system. In EHEC, almost all gsp genes have been lost during evolution, leaving only the gspO fragment encoding the prepilin peptidase catalytic domain. Additionally, EHEC strains have the gspO homologue etpO within a plsmid-encoded type 2 secretion system cluster. This gene is also a part of the generator of the major pilin PilA.
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==Characterization==
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We first uses the bacteria DH5a as the template and obtain the DNA fragment of 12 genes. We verified the practicability of the generator through the western blot of pilA in vivo which confirmed the usage of the generator because the PilA is able to display outside the cell.
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<partinfo>BBa_K3552000 parameters</partinfo>
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Revision as of 03:47, 26 October 2020


ppdA-ppdB-ygdB-ppdC-gspO(gene from DH5a)

Prepilin peptidase-dependent protein A, a minor pilin. A bitopic inner membrane protein located in the inner membrane, with the length of 471 bp/156 aa. prepilin peptidase-dependent protein B, a minor pilin. a bitopic inner membrane protein located in the cytosol of the inner membrane, with the length of 564 bp/ 187 aa. YgdB is a domain-containing protein with a length of 408 bp/135 aa. prepilin peptidase-dependent protein C, a bitopic inner membrane protein located in the inner membrane, with the length of 324 bp/ 107 aa. GspO plays a role in type II pseudopili formation by removing the leader sequence from substrate proteins and subsequently methylating the alpha-amino group of the newly exposed N-terminal phenylalanine. Substrates include proteins required for biogenesis of the type II general secretory apparatus. This part is in the part collection where we have 13 genes that code for the proteins.

The part collection includes: Parts that are different kinds of type 4 pilus: BBa_K3552003 BBa_K3552004 BBa_K3552005 BBa_K3552006 BBa_K3552007 BBa_K3552008 BBa_K3552018 BBa_K3552019 BBa_K3552020 BBa_K3552021 BBa_K3552022 BBa_K3552023 BBa_K3552024 BBa_K3552025 BBa_K3552026 BBa_K3552027 BBa_K3552028 BBa_K3552029

Our part collection can instruct other teams to designed new rechargeable pilus and substitution of different major pilin.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 15
    Illegal AgeI site found at 784
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1790

Usage and Biology

The ppdAB-ygdB-ppdC operon encodes the four minor pilins. These minor pilins and their homologues in the family contain two conserved Cys pairs at similar positions. We used the ppd genes from the EHEC strain EDL933 as building blocks to reconstitute an artificial T4P assembly operon in non-pathogenic E.coli, DH5a. Neither deletion of ppdC nor overproduction of ppdABC results in defects in chemotais or flagellar motility. This gene is predicted to be an inner membrane protein. The gene gspO, one of the two prepilin peptidase genes in E.coli K-12, is the last gene of the gsp operon encoding the chitinase-specific type 2 secretion system. In EHEC, almost all gsp genes have been lost during evolution, leaving only the gspO fragment encoding the prepilin peptidase catalytic domain. Additionally, EHEC strains have the gspO homologue etpO within a plsmid-encoded type 2 secretion system cluster. This gene is also a part of the generator of the major pilin PilA.

Characterization

We first uses the bacteria DH5a as the template and obtain the DNA fragment of 12 genes. We verified the practicability of the generator through the western blot of pilA in vivo which confirmed the usage of the generator because the PilA is able to display outside the cell.