Part:BBa_K4607007
Expression cassette for LysK-ABD-SH3B30 protein
Description
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), 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_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 ABD-endolysin lifetime is 30 hours [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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 938
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 938
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 938
Illegal XbaI site found at 96
Illegal SpeI site found at 127 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
As a brief contextualization, bovine mastitis is the result of the infection of the bovine mammary glands caused by pathogenic microorganisms, mainly gram-positive and negative bacteria. This disease reduces milk quality production to a great extent and produces painful damage to the bovine. The main treatment for mastitis is the use of diverse antibiotics, therefore the overuse and misuse of them have caused a real problem in the development of multidrug-resistant pathogens [6]. Our team has conducted an extensive investigation to find an alternative treatment for bovine mastitis without risking the environment.
To design our proposal of a novel non-antibiotic treatment against the losses of milk and bovine, capable of decreasing the effects on the milk industry and their consequences for the nutrition of the Mexican population, we took into account the CHAPk domain from the LysK of the bacteriophage K. This domain has an efficient catalytic activity against S. aureus strains, including the Methicillin-Resistant strains [7].
The principle behind the endolysin mechanism relies on the K bacteriophage. It is composed of three domains. For the design of a novel antimicrobial enzyme, the CHAPk domain from the K bacteriophage was selected for its 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 high efficiency. When the CHAPk domain is cloned as a truncated enzyme, which means that the endolysin is cloned without its other 2 domains: amidase-2 and cell-wall binding domain SH3b; it overexpresses as a soluble protein and has twice the activity of the native protein [7].
The endolysin LysK from the bacteriophage K has a CHAPk region with the ability to degrade the cell wall of antibiotic-resistant strains of Staphylococcus aureus [1] [2]. The function of a bacteriophage is to infect specific bacteria, in this case S. aureus, in order to kill them. Once the bacteria is infected and the virions are mature, they release holins, which are enzymes that create pores in the inner cell membrane. Endolysins now have access to the cell wall, so they can degrade it. Endolysins have a lytic activity for the purpose of setting free the phage progeny to continue infecting other cells [8].
The main purpose of the albumin binding domain (ABD) is to counteract the problems related to the brief in vivo time life of the endolysins. These domains have the capacity to increase the lifetime of antibodies, proteins, and enzymes through the incorporation of their sequences into the fusion protein. 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 best results have been achieved with the following conformation: CHAP domain-ABD-SH3 domain [9].
We also considered the domain SH3 from B30 bacteriophage that is capable of recognizing a wide spectrum of the pathogenic bacteria that cause bovine mastitis, including Streptococcus agalactiae, Streptococcus uberis, and Staphylococcus aureus. This characteristic results beneficial for the development of the treatment, considering that the intramammary infection caused by Streptococcus agalactiae often leads to subclinical mastitis, which can result in clinical mastitis [10].
A bacteriophage is a virus that targets a specific bacterial host and lyses its surface in order to degrade it at the end of its reproductive cycle. Endolysins have at least two domains: the N-terminal enzymatic activity domain for the lysis of the cell wall and the cell-wall binding domain (CBD) to attach to the cell wall of a specific host. If the CBD is fused with other domains of endolysins it can be used to attack particular bacteria in species or strain level [11] [12].
We intend to use the LysK-ABD-SH3B30 fused protein as a novel non-antibiotic proposal for fighting the bovine mastitis disease, specially the following pathogenic microbiota: Streptococcus uberis, Staphylococcus aureus, and Streptococcus agalactiae, which is completely safe for the host [4]. 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 against pathogenic bacteria but shows no toxic effects on mammalian cells.
Plasmid Construction and Analysis
To design this coding sequence, we have been evaluated different protein sequences through a protein structural model analysis tool, Alpha Fold 2, in order to identify the original functional protein structures and compare them with our final designs, ensuring their enzymatic activity. In the case of LysK-ABD-SH3B30 (Figure 2), we realized that the addition of the X6 HisTag (c) didn’t affect the protein’s functionality, and in comparison between the original LysK (a) and LysK-ABD-SH3B0 (d) sequences, no issue was encountered.
Golden Gate Assembly and identification
From the iGem 2023 plate, BioBrick™ BBa_J435330 (plate 2, well A15) was resuspended, which was then transformed into E. coli 5-alpha using heat shock and subsequently extracted and purified with the assistance of the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™, Lot #01260760). This part was chosen as the backbone due to its high replication origin, dual antibiotic resistance (kanamycin and ampicillin), and compatibility with the Golden Gate assembly method. Once the BBa_J435330 backbone was extracted and purified, Golden Gate assembly reactions were performed using the BsaI enzyme (NEB E1601, Lot #10182427) to insert each of the transcriptional units into the BBa_J435330 backbone.
After the Golden Gate assembly reaction, a bacterial transformation into E. coli 5-alpha was performed, followed by plating on LB agar with kanamycin (50 µg/mL). However, colonies were obtained both on the plates seeded with the transformation and in the negative controls. This led us to suspect that the plates from the Golden Gate reaction might contain a mixture of colonies transformed with the target construct and others transformed with the backbone alone, making them indistinguishable by visual inspection.
To confirm this, re-plating of several colonies from the same plate was conducted onto other plates with kanamycin and ampicillin. This was done to take advantage of the dual antibiotic resistance feature present in the utilized backbone. A colony from the reaction-seeded plate was selected and then plated onto two separate plates: one with kanamycin (50 µg/mL) and another with ampicillin (100 µg/mL). The same identifying number was assigned to both antibiotics.
The following day, observation revealed that some colonies grew on both antibiotics, while specific colonies only grew on kanamycin (50 µg/mL) and not on ampicillin (100 µg/mL). This suggested that these colonies might have been transformed with a construct carrying resistance only to kanamycin (50 µg/mL) and not to ampicillin. This led us to suspect that our construct might indeed be present in these colonies.
To verify this, a colony that exclusively exhibited kanamycin resistance (50 µg/mL) was selected. DNA plasmid extraction and purification were performed using the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen™, Lot #01260760). Subsequently, enzymatic digestion was carried out to linearize the DNA, followed by agarose gel electrophoresis for result visualization.
Using SnapGene® software, a simulation of enzymatic digestion was conducted on the constructs to identify potential enzymes for linearization. Subsequently, in the laboratory, the digestion reaction LysK-ABD-SH3B30 was performed using the SmaI restriction enzyme (Figure 3).
Protein Induction Kinetic
To identify the best protein induction parameters, we performed protein induction kinetics. Based on previous studies, we identify that the best temperature and time conditions were about 6 hours at 37° C. Related to the IPTG concentration, the experiments were performed at 0.2 mM, 0.5 mM, and 1 mM, for all of our transformants. The inductions were performed with the E. coli BL21 (DE3) transformed with the expression plasmids obtained from the Golden Gate Assembly.
Protein induction kinetics were performed at 37° C, for six hours at maximum agitation in an orbital shaker. Five treatments were included: Negative control with E. coli BL21 (DE3), negative control with E. coli BL21 (DE3) transformed with LysCSA13 without IPTG induction, and three E. coli BL21 (DE3) transformed with the endolysin induced at three different concentrations of IPTG, 0.2 mM, 0.5 mM, and 1 mM. The samples were collected by hour, harvesting 200 μL, by each treatment.
To identify the best induction parameters, a SDS-PAGE was performed. Considering that the molecular weight of LysK-ABD-SH3B30 was 28.437 kDa, the concentrations of the SDS-PAGE phases were 12% and 6%, for separator and concentrator phases respectively. The best parameters were IPTG at 0.2 nM and 5 hours for LysK-ABD-SH3B30 (Figure 4).
Massive Protein Induction
Once the best protein induction parameters were established, a new SDS-PAGE was performed to assess the protein expression site in the cell. Our endolysin were inducted at 0.2 mM IPTG, for six hours at 37° C. We obtained a massive protein expression after pellets were harvested. To evaluate the expression site, the reagent B-PER extracted the intracellular proteins, liberating the soluble and insoluble proteins. For the LysK-ABD-SH3B30 an soluble fraction has been demonstrated (Figure 5).
References
[1] Haddad Kashani, H., Schmelcher, M., Sabzalipoor, H., Seyed Hosseini, E., & Moniri, R. (2018). Recombinant endolysins as potential therapeutics against antibiotic-resistant Staphylococcus aureus: current status of research and novel delivery strategies. Clinical microbiology reviews, 31(1), 10-1128. https://doi.org/10.1128/cmr.00071-17
[2] Filatova, L. Y., Donovan, D. M., Ishnazarova, N. T., Foster-Frey, J. A., Becker, S. C., Pugachev, V. G., Dmitrieva, N. F., & Klyachko, N. L. (2016). A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: a study of both kinetics of inactivation and specifics of interaction with anionic polymers. Applied biochemistry and biotechnology, 180, 544-557. https://doi.org/10.1007/s12010-016-2115-7
[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.2018.02927
[4] Jarábková, V., Tišáková, L., Benešík, M., & Godány, A. (2021). SH3 binding domains from phage endolysins: how to use them for detection of gram-positive pathogens. Journal of Microbiology, Biotechnology and Food Sciences, 2021, 1215-1220. https://doi.org/10.15414/jmbfs.2020.9.6.1215-1220
[5] Lade, H., & Kim, J.-S. (2021). Bacterial Targets of Antibiotics in Methicillin-Resistant Staphylococcus aureus. Antibiotics, 10(4), 398. https://doi.org/10.3390/antibiotics10040398
[6] World Health Organization. (2021, November 17). Antimicrobial resistance. Who.int; World Health Organization: WHO. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance
[7] 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
[8] Gutiérrez, D., Fernández, L., Rodríguez, A., & García, P. (2018). Are phage lytic proteins the secret weapon to kill Staphylococcus aureus?. MBio, 9(1), 10-1128. https://doi.org/10.1128/mbio.01923-17
[9] Schmelcher, M., Powell, A. M., Becker, S. C., Camp, M. J., & Donovan, D. M. (2012). Chimeric Phage Lysins Act Synergistically with Lysostaphin To Kill Mastitis-Causing Staphylococcus aureus in Murine Mammary Glands. Applied and Environmental Microbiology, 78(7), 2297–2305. https://doi.org/10.1128/aem.07050-11
[10] Tong, J., Zhang, H., Zhang, Y., Xiong, B., & Jiang, L. (2019). Microbiome and metabolome analyses of milk from dairy cows with subclinical streptococcus agalactiae mastitis—potential biomarkers. Frontiers in microbiology, 10, 2547. https://doi.org/10.3389/fmicb.2019.02547
[11] Broendum, S. S., Buckle, A. M., & McGowan, S. (2018). Catalytic diversity and cell wall binding repeats in the phage‐encoded endolysins. Molecular microbiology, 110(6), 879-896. https://doi.org/10.1111/mmi.14134
[12] Cho, J. H., Kwon, J. G., O'Sullivan, D. J., Ryu, S., & Lee, J. H. (2021). Development of an endolysin enzyme and its cell wall–binding domain protein and their applications for biocontrol and rapid detection of Clostridium perfringens in food. Food Chemistry, 345, 128562. https://doi.org/10.1016/j.foodchem.2020.128562
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