Difference between revisions of "Part:BBa K1659000"

 
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<partinfo>BBa_K1659000 short</partinfo>
 
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This part contains the sequence for the ''Pseudomonas''-selective microbial lysis protein Art-175.
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===Usage and Biology===
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We have also made several parts with Art-175 fused to different secretion signal sequences:
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<html>
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    <table class="gridtable">
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        <tr>
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            <th>Secretion Tag</th>
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            <th>Part Number</th>
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        </tr>
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        <tr>
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            <td>None</td>
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            <td><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659000">BBa_K1659000</a></td>
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        </tr>
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        <tr>
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            <td>Flagellin 26-47 peptide segment</td>
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            <td><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659001">BBa_K1659001</a></td>
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        </tr>
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        <tr>
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            <td>DsbA</td>
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            <td><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659002">BBa_K1659002</a></td>
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        </tr>
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        <tr>
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            <td>YebF</td>
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            <td><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003">BBa_K1659003</a></td>
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        </tr>
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    </table>
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</html>
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===Biology===
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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. Endolysins have a degree of specificity in terms of of the peptidoglycan chemotype which they can break down by means of the structural selectivity of their enzymatically-active domain (EAD) or cell wall binding domain (CBD).
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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.
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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 against pathogenic ''P. aeruginosa'' strains PAO1 and PA14, being able to kill even persister cells effectively as it does not require active bacterial metabolism to exert its lytic activity [1].
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===Usage===
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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 an optimized strain of artilysin-producing ''E. coli'' from which we can purify Art-175 following cell lysis. We wish to compare unmodified Art-175 against its secretion-tagged cogeners which we have designed both in terms of antimicrobial activity as well as level of expression.
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===References===
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[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
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border-style: solid;
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===Functional Parameters===
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Revision as of 18:51, 30 August 2015

Artilysin Art-175, an antibacterial fusion protein


This part contains the sequence for the Pseudomonas-selective microbial lysis protein Art-175.

We have also made several parts with Art-175 fused to different secretion signal sequences:


Secretion Tag Part Number
None BBa_K1659000
Flagellin 26-47 peptide segment BBa_K1659001
DsbA BBa_K1659002
YebF BBa_K1659003


Biology

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. Endolysins have a degree of specificity in terms of of the peptidoglycan chemotype which they can break down by means of the structural selectivity of their enzymatically-active domain (EAD) or cell wall binding domain (CBD).

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 against pathogenic P. aeruginosa strains PAO1 and PA14, being able to kill even persister cells effectively as it does not require active bacterial metabolism to exert its lytic activity [1].


Usage

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 an optimized strain of artilysin-producing E. coli from which we can purify Art-175 following cell lysis. We wish to compare unmodified Art-175 against its secretion-tagged cogeners which we have designed both in terms of antimicrobial activity as well as level of expression.


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