Coding

Part:BBa_K4607001

Designed by: Axel Rojero   Group: iGEM23_Tec-Chihuahua   (2023-07-21)
Revision as of 06:30, 21 July 2023 by Axelrohz24 (Talk | contribs)


LysK-ABD-SH3B30

Figure 1.LysK-ABD-SH3B30 protein diagram.

This biobrick is a fusion protein based on the endolysin (Lys) from Staphylococcus aureus bacteriophage K [1], the albumin binding domain (ABD) from streptococcal protein G [2], and the SH3 domain from the Streptococcus agalactiae, Streptococcus uberis y S. aureus bacteriophage B30[3]. Its purpose is to recognize the S. aureus, Streptococcus. agalactiae, Streptococcus uberis cell wall, and degrade it. The part is adapted to the Golden Gate cloning method.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 415
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 328
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

The endolysin Lys from the K bacteriophage, is composed of three domains. For the design of a novel antimicrobial enzyme, the CHAPk domain from the K bacteriophage was selected for their ability to cleave between the D-alanine and the first glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the peptidoglycan, with a high efficiency [1]. To increase the sensitivity of the enzyme for pathogenic bacteria, specifically Streptococcus uberis, Staphylococcus aureus, and Streptococcus agalactiae, the SH3 domain from the B30 bacteriophage was selected, which makes it completely safe for the host [3].

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 a length of 786 bp and contains an extra region, the albumin binding domain, that causes an important increase in the life-time of the fusion protein [2].

Table 1.LysK-ABD-SH3B30 protein parameters.

Results

Figure 2. 3D structure of the LysK-ABD-SH3B30 protein, obtained with AlphaFold2.

References

[1] Sanz-Gaitero, M., Keary, R., Garcia-Doval, C., Coffey, A., & van Raaij, M. J. (2013). Crystallization of the CHAP domain of the endolysin from Staphylococcus aureusbacteriophage K. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 69(12), 1393–1396. https://doi.org/10.1107/s1744309113030133

[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

[3] Jarábková, V., Tišáková, L., Benešík, M., & Godány, A. (2020). SH3 BINDING DOMAINS FROM PHAGE ENDOLYSINS: HOW TO USE THEM FOR DETECTION OF GRAMPOSITIVE PATHOGENS. Www.muni.cz, 9(6). https://www.muni.cz/vyzkum/publikace/1674660

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