Difference between revisions of "Part:BBa K4607007"

Revision as of 23:23, 27 July 2023

Expression cassette for LysK-ABD-SH3B30 protein

Figure 1. Expression cassette for LysK-ABD-SH3B30 protein diagram.

Expression cassette for LysK-ABD-SH3B30 protein: Fusion endolysin against Staphylococcus aureus,Streptococcus agalactiae, and Streptococcus uberis bacteria who causes bovine mastitis

This part contains the linear sequence of the LysK-ABD-SH3B30 (BBa_K4607001) nucleotide optimized for E. coli. It incorporates some of the most efficient biobricks as described below: 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_K4607001 that codifies for a fusion protein of the CHAP domain from the Lys of the bacteriophage K, with the ability to degrade the cell wall of antibiotic-resistant strains of Staphylococcus aureus [1] [2]; the albumin binding domain (ABD) from streptococcal protein G that is capable of increasing antibody, protein, and enzyme lifetimes; and the SH3 domain from the bacteriophage B30, which binds to the cell-wall of Streptococcus agalactiae, Streptococcus uberis, and Staphylococcus aureus. The ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. The domain consists of an affinity-maturated variant of the streptococcal protein G, which has been used for LysK expression with results of up to 34 hours in increasing the lifetime of the protein in mice [3]. The principle of the mechanism of the SH3B30 domain is to recognize and bind to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the S. aureus, S. agalactiae and S. uberis peptidoglycan, activating the catalytic domain [4][5]. The enzyme has a length of 262 amino acids and a molecular weight of 28.437 kDa. The average endolysin lifetime is 20 minutes [3]. The part is adapted to the Golden Gate cloning method. This part also contains a x6 HisTag in the C-terminal site, to facilitate its purification process.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 938
Illegal XbaI site found at 96
Illegal SpeI site found at 127
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 938
Illegal NheI site found at 552
Illegal SpeI site found at 127
Illegal NotI site found at 1080
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 938
Illegal BglII site found at 30
Illegal BamHI site found at 465
Illegal BamHI site found at 932
Illegal XhoI site found at 1089
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 938
Illegal XbaI site found at 96
Illegal SpeI site found at 127
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 938
Illegal XbaI site found at 96
Illegal SpeI site found at 127
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Biobrick selection

LysK-ABD-SH3B30 Fusion Design

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 [2].

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 [3].

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 aureus bacteriophage K. Acta Crystallographica Section F Structural Biology and Crystallization Communications, 69(12), 1393–1396. https://doi.org/10.1107/s1744309113030133

[2] 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

[3] 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

@@ Line 12: / Line 12: @@
-This part contains the linear sequence of the LysK-ABD-SH3B30 (<partinfo>BBa_K4607001</partinfo>) nucleotide optimized for <i>E. coli</i>. It incorporates some of the most efficient biobricks as described below: the T7 promoter with LacO regulations  (<partinfo>BBa_J435350</partinfo>), the medium strength RBS (<partinfo>BBa_Z0262</partinfo>), the triple terminator  (<partinfo>BBa_J435371</partinfo>), and the high copy pUC ori /Kan R backbone (<partinfo>BBa_J435330</partinfo>). It also contains the <partinfo>BBa_K4607001</partinfo> that codifies for the fused endolysin from the CHAPk K bacteriophage, with a very efficient catalytic activity, the albumin binding domain that increases its lifetime, the SH3 B30 bacteriophage domain, and the x6 HisTag for their posterior purification.
+This part contains the linear sequence of the LysK-ABD-SH3B30 (<partinfo>BBa_K4607001</partinfo>) nucleotide optimized for <i>E. coli</i>. It incorporates some of the most efficient biobricks as described below: the T7 promoter with LacO regulations  (<partinfo>BBa_J435350</partinfo>), the medium strength RBS (<partinfo>BBa_Z0262</partinfo>), the triple terminator  (<partinfo>BBa_J435371</partinfo>), and the high copy pUC ori /Kan R backbone (<partinfo>BBa_J435330</partinfo>). It also contains the <partinfo>BBa_K4607001</partinfo> that codifies for a fusion protein of the CHAP domain from the Lys of the bacteriophage K, with the ability to degrade the cell wall of antibiotic-resistant strains of <i>Staphylococcus aureus</i> [1] [2]; the albumin binding domain (ABD) from streptococcal protein G that is capable of increasing antibody, protein, and enzyme lifetimes; and the SH3 domain from the bacteriophage B30, which binds to the cell-wall of <i>Streptococcus agalactiae</i>, <i>Streptococcus uberis</i>, and <i>Staphylococcus aureus</i>. The ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. The domain consists of an affinity-maturated variant of the streptococcal protein G, which has been used for LysK expression with results of up to 34 hours in increasing the lifetime of the protein in mice [3]. The principle of the mechanism of the SH3B30 domain is to recognize and bind to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the <i>S. aureus</i>, <i>S. agalactiae</i> and <i>S. uberis </i>peptidoglycan, activating the catalytic domain [4][5]. The enzyme has a length of 262 amino acids and a molecular weight of 28.437 kDa. The average endolysin lifetime is 20 minutes [3]. The part is adapted to the Golden Gate cloning method. This part also contains a x6 HisTag in the C-terminal site, to facilitate its purification process.
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 ===<span class='h3bb'><b>Sequence and Features</b></span>===
 <partinfo>BBa_K4607007 SequenceAndFeatures</partinfo>