Difference between revisions of "Part:BBa K4607006"

 
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This part contains the linear sequence of the LysCSA13-ABD nucleotide optimized for E. coli. It incorporates some of the most efficient biobricks as described bellow: the T7 promoter with LacO regulations BBa_J435350, the medium strength RBS BBa_Z0262, the triple terminator BBa_J435371, and the high copy pUC ori /Kan R backbone BBa_J435330. It also contains the BBa_K4607000 that codifies for the fused endolysin from the CSA13 bacteriophage, with a very efficient catalytic activity, the albumin binding domain that increases its lifetime, and the x6 HisTag for their posterior purification. Additionally, it has a TEV cleavage site for the removal of the purification tag.
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<center><b>Figure 1.</b> LysCSA13-ABD protein diagram.</center>
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This part contains the linear sequence of the LysCSA13-ABD nucleotide optimized for <i>E. coli</i>. It incorporates some of the most efficient biobricks as described bellow: the T7 promoter with LacO regulations BBa_J435350, the medium strength RBS BBa_Z0262, the triple terminator BBa_J435371, and the high copy pUC ori /Kan R backbone BBa_J435330. It also contains the BBa_K4607000 that codifies for the fused endolysin from the CSA13 bacteriophage, with a very efficient catalytic activity, the albumin binding domain that increases its lifetime, and the x6 HisTag for their posterior purification. Additionally, it has a TEV cleavage site for the removal of the purification tag.
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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===<span class='h3bb'><b>Sequence and Features</b></span>===
 
<partinfo>BBa_K4607006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4607006 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
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<div style="text-align:justify;">
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The endolysin Lys from the <i>Staphylococcus aureus virulent bacteriophage CSA1</i>, is composed of two domains. The bacteriophage CSA13 CHAP domain has excellent catalytic activity, up to 90%, degrading almost 15 strains of <i>Staphylococcus</i> including methicillin-resistant strains (MRSA). As with many of the endolysins, it cleaves to the cell wall by disrupting the peptidoglycan that composes the bacterial cell; for this to be possible, the bacteriophage CSA13 SH3 domain recognizes and binds to the glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the peptidoglycan, which makes it completely safe for the host [1].
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The use of enzybiotics represents an alternative to the misuse of antibiotics without loss of efficiency; it is a novel and environmentally friendly process. It supplies antibacterial protection to pathogenic bacteria but shows no toxic effects on mammalian cells. Our protein has an extra region, the albumin binding domain, that causes an important increase in the life-time of the fusion protein [2].
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<center><b>Table 1.</b> LysCSA13-ABD protein parameters.</center>
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===Results===
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<center><b>Figure 2.</b> 3D structure of the LysCSA13-ABD protein, obtained with AlphaFold2.</center>
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===References===
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[1] Cha, Y., Son, B., & Ryu, S. (2019). Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13. Food Microbiology, 84, 103245. https://doi.org/10.1016/j.fm.2019.103245
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[2] Seijsing, J., Sobieraj, A. M., Keller, N., Shen, Y., Zinkernagel, A. S., Loessner, M. J., & Schmelcher, M. (2018). Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.029
  
 
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Revision as of 07:34, 21 July 2023


Expression cassette for LysCSA13-ABD protein

Figure 1. LysCSA13-ABD protein diagram.

This part contains the linear sequence of the LysCSA13-ABD nucleotide optimized for E. coli. It incorporates some of the most efficient biobricks as described bellow: the T7 promoter with LacO regulations BBa_J435350, the medium strength RBS BBa_Z0262, the triple terminator BBa_J435371, and the high copy pUC ori /Kan R backbone BBa_J435330. It also contains the BBa_K4607000 that codifies for the fused endolysin from the CSA13 bacteriophage, with a very efficient catalytic activity, the albumin binding domain that increases its lifetime, and the x6 HisTag for their posterior purification. Additionally, it has a TEV cleavage site for the removal of the purification tag.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1100
    Illegal XbaI site found at 96
    Illegal SpeI site found at 127
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1100
    Illegal SpeI site found at 127
    Illegal NotI site found at 1242
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1100
    Illegal BglII site found at 30
    Illegal BamHI site found at 1094
    Illegal XhoI site found at 1251
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1100
    Illegal XbaI site found at 96
    Illegal SpeI site found at 127
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1100
    Illegal XbaI site found at 96
    Illegal SpeI site found at 127
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

The endolysin Lys from the Staphylococcus aureus virulent bacteriophage CSA1, is composed of two domains. The bacteriophage CSA13 CHAP domain has excellent catalytic activity, up to 90%, degrading almost 15 strains of Staphylococcus including methicillin-resistant strains (MRSA). As with many of the endolysins, it cleaves to the cell wall by disrupting the peptidoglycan that composes the bacterial cell; for this to be possible, the bacteriophage CSA13 SH3 domain recognizes and binds to the glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the peptidoglycan, which makes it completely safe for the host [1].

The use of enzybiotics represents an alternative to the misuse of antibiotics without loss of efficiency; it is a novel and environmentally friendly process. It supplies antibacterial protection to pathogenic bacteria but shows no toxic effects on mammalian cells. Our protein has an extra region, the albumin binding domain, that causes an important increase in the life-time of the fusion protein [2].

Table 1. LysCSA13-ABD protein parameters.

Results

Figure 2. 3D structure of the LysCSA13-ABD protein, obtained with AlphaFold2.

References

[1] Cha, Y., Son, B., & Ryu, S. (2019). Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13. Food Microbiology, 84, 103245. https://doi.org/10.1016/j.fm.2019.103245

[2] Seijsing, J., Sobieraj, A. M., Keller, N., Shen, Y., Zinkernagel, A. S., Loessner, M. J., & Schmelcher, M. (2018). Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.029