Difference between revisions of "Part:BBa K1659001"

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This part contains the sequence for the ''Pseudomonas''-selective microbial lysis protein Art-175 with a flagellar secretion signal peptide fused to its N-terminus and a Hisx6 tag fused to its C-terminus.
 
This part contains the sequence for the ''Pseudomonas''-selective microbial lysis protein Art-175 with a flagellar secretion signal peptide fused to its N-terminus and a Hisx6 tag fused to its C-terminus.
  
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===Usage===
 
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To facilitate the secretion of Art-175 via the flagellar export system, we fused the flagellin 26-47 secretion signal to the N-terminus of Art-175. A hexahistidine tag is also attached onto the C-terminus of the composite to allow for easy purification of the expressed protein via metal-affinity column chromatography.
 
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By transforming this coding sequence in a commercial pBAD expression vector into our host organisms of choice, ''E. coli'' MG1655, ''E. coli'' RP437 ∆FliC, and ''E. coli'' DH5α, we hope to create a strain of ''Pseudomonas''-killing ''E. coli'' that when used in conjunction with our other DNase-secreting strain can effectively eradicate biofilm-protected ''P. putida'' and ''P. aeruginosa''.
 
  
 
===Biology===
 
===Biology===
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Flagellin are the constituent subunits of the helical filament substructure of bacterial flagella. In the flagellar-building process, flagellin are exported out of the cell sequentially by the flagellum-specific export apparatus. F. Vonderviszt et al. demonstrated through their work that the signal sequence responsible for allowing the flagellar export system to identify and export ''Salmonella'' flagellin is its 26-47 amino acid residue segment [2].
 
Flagellin are the constituent subunits of the helical filament substructure of bacterial flagella. In the flagellar-building process, flagellin are exported out of the cell sequentially by the flagellum-specific export apparatus. F. Vonderviszt et al. demonstrated through their work that the signal sequence responsible for allowing the flagellar export system to identify and export ''Salmonella'' flagellin is its 26-47 amino acid residue segment [2].
 +
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===Usage===
 +
 +
To facilitate the secretion of Art-175 via the flagellar export system, we fused the flagellin 26-47 secretion signal to the N-terminus of Art-175. A hexahistidine tag is also attached onto the C-terminus of the composite to allow for easy purification of the expressed protein via metal-affinity column chromatography.
 +
 +
By transforming this coding sequence in a commercial pBAD expression vector into our host organisms of choice, ''E. coli'' MG1655, ''E. coli'' RP437 ∆FliC, and ''E. coli'' DH5α, we hope to create a strain of ''Pseudomonas''-killing ''E. coli'' that when used in conjunction with our other DNase-secreting strain can effectively eradicate biofilm-protected ''P. putida'' and ''P. aeruginosa''.
 +
 +
  
 
===Sequence and Features===
 
===Sequence and Features===
  
 
<partinfo>BBa_K1659001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1659001 SequenceAndFeatures</partinfo>
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===References===
 
===References===

Revision as of 10:08, 27 August 2015

Artilysin Art-175 fused at N-terminal with flagellin 26-47 peptide segment

This part contains the sequence for the Pseudomonas-selective microbial lysis protein Art-175 with a flagellar secretion signal peptide fused to its N-terminus and a Hisx6 tag fused to its C-terminus.


Biology

BBa_K1659001 is a composite of artilysin Art-175 with the 26-47 peptide segment of Salmonella typhimurium flagellin:


1. Art-175

Artilysins are an exciting class of enzyme-based antibacterials. Their name is derived from "artificial endolysin" and they exploit the lytic power of bacteriophage-encoded endolyins. Endolysins are peptidoglycan hydrolases produced at the end of the lytic cycle that pass through the cytoplasmic membrane, degrade the peptidoglycan layer and cause the osmotic lysis of the infected bacterial cell, thus liberating the progeny. Purified endolysins have been used to kill Gram-positive pathogens. Gram-negative bacteria, however, have a protective outer membrane containing lipopolysaccharide (LPS) that serves as a barrier against the peptidoglycan hydrolytic activity of endolysins from the outside.

To overcome this problem, selected polycationic or amphipathic peptides that locally destabilize the LPS layer can be covalently fused to endolysins to transport them past the outer membrane to reach the peptidoglycan layer. Biers et al. fused the sheep myeloid antimicrobial peptide (SMAP-29), which introduces transient cracks in the outer membrane by means of interaction with cationic binding sites combined with hydrophobic disruption of barrier function, to the N-terminus of the endolysin KZ144 to create Artilysin Art-175. Art-175 has been shown to be a highly potent antibacterial that acts in minutes to kill pathogenic P. aeruginosa strains PAO1 and PA14 [1].


2. Flagellin 26-47 secretion signal

Flagellin are the constituent subunits of the helical filament substructure of bacterial flagella. In the flagellar-building process, flagellin are exported out of the cell sequentially by the flagellum-specific export apparatus. F. Vonderviszt et al. demonstrated through their work that the signal sequence responsible for allowing the flagellar export system to identify and export Salmonella flagellin is its 26-47 amino acid residue segment [2].


Usage

To facilitate the secretion of Art-175 via the flagellar export system, we fused the flagellin 26-47 secretion signal to the N-terminus of Art-175. A hexahistidine tag is also attached onto the C-terminus of the composite to allow for easy purification of the expressed protein via metal-affinity column chromatography.

By transforming this coding sequence in a commercial pBAD expression vector into our host organisms of choice, E. coli MG1655, E. coli RP437 ∆FliC, and E. coli DH5α, we hope to create a strain of Pseudomonas-killing E. coli that when used in conjunction with our other DNase-secreting strain can effectively eradicate biofilm-protected P. putida and P. aeruginosa.


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 AgeI site found at 525
    Illegal AgeI site found at 724
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J. P., Defraine, V., … Lavigne, R. (2014). Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 58(7), 3774–3784. http://doi.org/10.1128/AAC.02668-14

[2] Vondervizst, F., Sajó, R., Dobó, J., & Závodszky, P. (2012). The Use of a Flagellar Export Signal for the Secretion of Recombinant Proteins in Salmonella. In: Recombinant Gene Expression - Reviews and Protocols, Methods in Molecular Biology, 824, 131-143. http://doi.org/10.1007/978-1-61779-433-9_6