Figure 1. LysCSA13-ABD protein diagram.

The biobrick consists of a fusion protein based on the endolysin Lys from Staphylococcus aureus's virulent bacteriophage CSA13 which is composed of three domains: the CHAP domain, with an excellent catalytic activity of up to 90%, degrading almost 15 strains of Staphylococcus, including methicillin-resistant strains (MRSA).¹ Secondly, the SH3 domain, which recognizes and binds to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the S. aureus peptidoglycan, activating the catalytic domain.² Finally, there's the albumin binding domain (ABD) from streptococcal protein G. The ABD is capable of increasing the antibody, protein, and enzyme lifetimes. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This section consists of an affinity-maturated variant of the streptococcal protein G which has been used for the expression of LysK in mice, increasing the protein's lifetime up to 34 hours.³ The part is adapted to the Golden Gate cloning method. Additionally, this part contains a TEV cleavage site for the removal of the 6xHis-Tag after the protein purification process. The enzyme has a length of 316 amino acids and a molecular weight of 35.098 kDa. It keeps its stability in a range of 4ºC to 37°C and a pH of 7 to 9, and it has extracellular protein expression. Other characteristics of the endolysin are that it requires the presence of calcium and manganese to reach its maximum catalytic activity. The average endolysin lifetime is about 30 hours.³

Figure 2. LysK-ABD-SH3B30 protein diagram.

The biobrick consists of the fusion 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;^4,5 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, increasing the protein's lifetime in mice for over 34 hours.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.^2,6 The enzyme has a length of 262 amino acids and a molecular weight of 28.437 kDa. The average ABD- endolysin lifetime is 30 hours.³ The part is adapted to the Golden Gate cloning method. This part also contains a 6xHis-Tag in the C-terminal site, to facilitate its purification process.

Figure 3. Albumin binding domain diagram.

The biobrick consists of the albumin binding domain (ABD) from streptococcal protein G and is capable of increasing the lifetimes of antibodies, proteins, and enzymes. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This part 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 domain has a length of 29 amino acids and a molecular weight of 3.2388 kDa.

Figure 4. SH3 domain from the B30 bacteriophage diagram.

The biobrick consists of the SH3 domain from the bacteriophage B30, which is capable of recognizing and binding to the cell wall of Streptococcus agalactiae, Streptococcus uberis, and Staphylococcus aureus. For this to be possible, the SH3B30 domain recognizes and binds 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. The enzyme has a length of 62 amino acids and a molecular weight of 7.162 kDa. It keeps its stability at a range of 4 to 37° C and pH from 7 to 8.2.⁶

Figure 5. Lys from the CSA13 bacteriophage diagram.

The biobrick consists of the endolysin Lys from the Staphylococcus aureus virulent bacteriophage CSA13, and it is composed of two domains: the CHAP domain, with an excellent catalytic activity of up to 90%, degrading almost 15 strains of Staphylococcus, including methicillin-resistant strains (MRSA)¹, and the SH3 domain, which recognizes and binds to the highly specific glycine of the pentaglycine cross-bridge glycosidic bond in the heteropolymer of the S. aureus peptidoglycan, activating the catalytic domain.² The enzyme has a length of 250 amino acids and a molecular weight of 28.36 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9, and it has extracellular protein expression. The average endolysin lifetime is about 20 minutes.³ The part is adapted to the Golden Gate cloning method.

Figure 6. CHAPk domain from the k bacteriophage diagram.

The biobrick consists of the CHAPk domain from the Lys of the bacteriophage K, with the ability to degrade the cell wall of antibiotic-resistant strains of Staphylococcus aureus.^4,5 The domain expresses in a soluble fraction and has twice the catalytic activity of the original enzyme. The principle of its catalytic activity is the cleavage between the first glycine of the pentaglycine cross-bridge and D-alanine. The CHAPk domain contains 125 amino acids and a molecular weight of 18.6 kDa. It loses its activity at a temperature of 42.5 °C, meaning it denatures, and works at a pH of 6.5 to 7.5.⁷

Figure 7. PCNP peptide diagram.

The biobrick consists of a polycationic nonapeptide (PCNP) which is capable of destabilizing the lipopolysaccharide and binding to gram-negative bateria. The principle of the PCNP is related to the ionic interactions between the phosphate groups, divalent cations, and hydrophobic lipids' stacking, where the PCNP acts as a destabilizing agent. This part has been fused with endolysins in order to increase their capability to lyse gram-negative bacteria, demonstrating that the addition of the PCNP allows the endolysin introduction into the bacteria's cell membrane, with supporting evidence when evaluated in Escherichia coli. The peptide has a length of 9 amino acids and is adapted with an additional flexible linker sequence in order to increase its possibility of being incorporated into new codifying sequences without affecting the functionality of other parts or their domains' requirements.⁸

Figure 8. CecA peptide diagram.

This biobrick consists of cecropin A (CecA), which was selected for its ability as an antimicrobial peptide. CecA has demonstrated excellent capacity for improving the endolysins antibacterial activity against gram-negative bacteria when it's incorporated in the N-terminal region. The principle behind CecA's antibacterial potential resides in its composition, which includes a cationic region that facilitates lipid interactions, favors a stronger ionic interaction, and finally degrades the cell wall by damaging bacterial inner membranes. CecA peptide has been evaluated in gram-negative bacteria as Escherichia coli. This part has a lenght of 41 amino acids.⁹

Figure 9. LysSS protein diagram.

This biobrick consists of the endolysin LysSS from the bacteriophage SS3e from Salmonella that has demonstrated antibacterial activity against gram-negative bacteria as Escherichia coli, and gram-negative bacteria such as Staphylococcus aureus including methicillin-resistant strains. In comparison with other endolysins, LysSS contains positive charges at the C-terminal region that destabilize the gram-negative cell membrane. This part has a length of 173 amino acids and a molecular weight of 18.56 kDa. LysSS has intracellular and insoluble protein expression. The average endolysin lifetime is about 30 hours. The part is adapted to the Golden Gate cloning method. ¹⁰

Figure 10. PCNP-CecA-LysSS fusion-protein diagram.

This biobrick consists of a fusion protein based on three main parts: the polycationic nonapeptide (PCNP), Cecropin A peptide, and the LysSS protein, all integrated with flexible linkers that ensure its functionality. The first part, PCNP is capable of destabilizing the lipopolysaccharide and binding to gram-negative bacteria. The principle of the PCNP is related to the ionic interactions between the phosphate groups, divalent cations, and hydrophobic lipids' stacking, where the PCNP acts as a destabilizing agent. This part has been fused with endolysins in order to increase their capability to lyse gram-negative bacteria, demonstrating that the addition of the PCNP allows the endolysin introduction into the bacteria's cell membrane. It has been evaluated in Escherichia coli.⁸ The second part is cecropin A (CecA), which was selected for its ability as an antimicrobial peptide. CecA has demonstrated excellent capacity for improving the endolysins antibacterial activity against gram-negative bacteria when it's incorporated in the N-terminal region. The principle behind CecA's antibacterial potential resides in its composition, which includes a cationic region that facilitates lipid interactions, favors a stronger ionic interaction, and finally degrades the cell wall by damaging bacterial inner membranes, as proven in Escherichia coli.⁹ And finally, the endolysin LysSS from the bacteriophage SS3e from Salmonella that has demonstrated antibacterial activity against gram-negative bacteria such as Escherichia coli, and gram-positive bacteria such as Staphylococcus aureus including methicillin-resistant strains. In comparison with other endolysins, LysSS contains positive charges at the C-terminal region that destabilize the gram-negative cell membrane. LysSS has intracellular and insoluble protein expression. The fusion protein PCNP-CecA-LysSS has a lenght of 227 amino acids and a molecular weight of 24.359 kDa.¹⁰

Fusion endolysin against S. aureus and other bacteria who cause bovine mastitis.

Figure 11. LysCSA13-ABD protein diagram.

This part contains the linear sequence of the LysCSA13-ABD (BBa_K4607000) 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, and the triple terminator BBa_J435371. For our plasmid construction, we used the high copy pUC ori/Kan R backbone BBa_J435330. It also contains the BBa_K4607000 that codifies for a fusion protein based on the endolysin Lys from Staphylococcus aureus virulent bacteriophage CSA13, which have the ability to lyse S. aureus with very specific targeting and an excellent catalytic activity^1,6,3.

Fusion endolysin against S. aureus.

Figure 12. LysCSA13-ABD protein diagram.

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 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, as well as, S. agalactiae and S. uberis.^4,5,2,6

Fusion artilysin against E. coli

Figure 13. PCNP-CecA-LysSS expression cassette diagram.

This part contains the linear sequence of the PCNP-CecA-LysSS nucleotide optimized for E. coli. It incorporates some of the most efficient biobricks as described below: the T7 promoter with LacO regulation, the medium strength RBS, and the triple terminator. For our plasmid construction, we used the high copy pUC ori /Kan R backbone. It also contains a fusion protein based on three main parts: the polycationic nonapeptide (PCNP), Cecropin A peptide, and the LysSS protein, all integrated with flexible linkers that assure its functionality.