Overview

AureoBos's general purpose is to develop a novel solution for bovine mastitis by approaching it from an unexplored side in Mexico: synthetic biology. Hence, our project's path starts in functional BioBricks. By analyzing various molecular mechanisms existing in nature, endolysins caught our attention because of their mechanism and specificity. By mimicking the endolysin's mechanism, our lab developed antimicrobial fusion proteins that fight four of the main microorganisms known to cause bovine mastitis: Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae. and Escherichia coli. Throughout this page you can find the bioparts used and designed to construct the expression cassettes that paved the way of this project.

Achievements

Round Zero: Plasmid Construction and Analysis

The initial round of experimentation encompassed the in silico design of our plasmids, their synthesis, assembly, transformation, and analysis of the constructs. This comprehensive phase integrates computational design with practical implementation, culminating in the evaluation of the resulting constructs. To design our endolysins coding sequences, we conducted an in-depth protein analysis. This helped us to identify the crucial domains required to produce highly effective and catalytically specific endolysins. The resulting sequences encode the following intein-fusion proteins:

Composite Parts

Expression cassette for LysCSA13-ABD protein

Fusion endolysin against S. aureus bacteria who causes bovine mastitis.

Figure 1. 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.^1,2,3

Expression cassette for LysK-ABD-SH3B30 protein

Fusion endolysin against S. aureus bacteria who causes bovine mastitis.

Figure 2. 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.^4,5,6,2

Expression cassette for PCNP-CecA-LysSS protein

Fusion artilysin against E. coli

Figure 3. 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.^7,8,9

The coding sequence (CDS) of the three recombinant intein-fusion proteins (LysK endolysin, LysCSA13, and LysSS) was in silico edited to incorporate BsaI enzyme recognition sites at the ends of the sequence. In the same way, each CDS design has been evaluated through the protein structural model analysis tool, Alpha Fold 2, to identify the original functional protein structures and compare them with our final designs, ensuring their enzymatic activity (Figure 4 - 6).A great quantity of protein chains can be predicted accurately by using the AlphaFold 1 model; this model has the possibility to predict protein structures from a dataset of 4446 protein complexes, without non-redundant interfaces. The AlphaFold 2.0 offers a very high accuracy in the Critical Assessment of Protein Structure Prediction, and by the 2021 the AlphaFold Protein Structure Database reach an unprecedented group of reliability completely accessible to the scientific community. Thanks to its algorithm it is possible to simulate three-dimensional models for the whole human proteome, and a high variety of proteins.^1–3 The best LysCSA13-ABD option was developed by identifying that the presence of 6x HisTag (c) affects the functional protein domains (Figure 4) in comparison to the original LysCSA13 (a) and LysCSA13-ABD (b) structures. To solve this problem, we added a TEV site to its posterior removal (d). In the case of LysK-ABD-SH3B30 (Figure 5), 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. Finally, (Figure 6) LysSS demonstrated that using short flexible linkers^4,5, any of the original peptide sequences (a), (b), or LysSS original protein sequence (c) would have been affected, so the final design (d) conserved its enzymatic activity. Additionally, we assured that all our final intein-fusion proteins kept the original proteins’ distribution with functional domains.^6-9

Parts were assembled in SnapGene® software using the Golden Gate method, employing the type IIS restriction enzyme BsaI. The transcriptional units (TUs) of each of our endolysins (TU LysK, TU LysCSA13, and TU LysSS) were synthesized by Twist Bioscience, making this possible by the sponsorship of 10 kb of FREE Twist Gene Fragments for iGEM teams. This genetic material was delivered lyophilized with a total of 1000 ng per tube, which were resuspended in 100 µL of TE to achieve a final concentration of 10 ng/mL for each of the transcriptional units. Subsequently, aliquots of each TU were prepared to proceed with the Golden Gate assembly reaction.

First Round: Golden Gate Assembly

Golden Gate Assembly Reaction

From the iGEM 2023 plate, the BioBrick™ BBa_J435330 (plate 2, well A15) was resuspended, which was transformed into E. coli 5-alpha by heat shock, and subsequently extracted and purified with 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 in order to take advantage of the dual antibiotic resistance feature present in the used 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 next day, observation revealed that some colonies grew on both antibiotics, while specific colonies only grew on kanamycin (50 µg/mL) and on ampicillin there wasn't any growth (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 exhibited only 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.

Evaluation of the resulting constructs

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 of the three constructs LysK, LysCSA13, and LysSS was performed using SmaI and EcoRI restriction enzymes. LysK was linearized by the SmaI enzyme, while both LysCSA13 and LysSS were linearized by EcoRI (Figure 8). All our constructions were according to the expected bands.^5,7,10,11

Figure 8. pDNA agarose gel electrophoresis (1%). (a) Lane 1. Quick-Load® Purple 1 kb Plus DNA Ladder. Lane 2. LysK digested with single-cut enzyme SmaI (2997 bp) pDNA. Lane 3. Negative control LysK digestion (2997 bp) pDNA. (b) Lane 4. Quick-Load® Purple 1 kb Plus DNA Ladder. Lane 5. LysCSA13 digested with single-cut enzyme EcoRI. Lane 6. Negative control LysCSA13 digestion (3159 bp) pDNA. (c) Lane 7. LysSS digested with single-cut enzyme EcoRI (2892 bp). Lane 8. Negative control LysSS digestion (2892 bp) pDNA. Lane 9. Quick-Load® Purple 1 kb Plus DNA Ladder.

Second Round: Protein Expression

The next step of our recombinant protein production process was grouped into the second round of experimentation. All the selection, optimization, and standardization of our protein induction protocols were performed as follows:

Protein production

Our constructs contain the biobrick BBa_J435350, a T7 promoter with LacO regulation regions. It was chosen because of its high transcription levels, and regulation activity. To induce the protein production of our E. coli BL21 (DE3) transformants, isopropyl β-D-1 thiogalactopyranoside (IPTG) was used as inducer for the LacO regulation regions, then the reagent makes the transformants available to synthetize our endolysins.

Protein induction kinetics

To identify the best protein induction parameters, protein induction kinetics were performed. Based on previous studies, we identified that the optimal 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 6 hours at maximum agitation in an orbital shaker. Five treatments were included for each endolysin: Negative control with E. coli BL21 (DE3), negative control with E. coli BL21 (DE3) transformed with the endolysin 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, obtaining 200 μL, by each treatment.

To identify the best induction parameters, a SDS-PAGE was performed. Considering that the molecular weight of LysCSA13-ABD was 35.098 kDa, and LysK-ABD-SH3B30 weights 28.437 kDa.^8,12,13 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 mM and 5 hours for LysCSA13-ABD, while LysK-ABD-SH3B30 requires 6 hours at the same IPTG concentration, results can be seen in Figures 6 & 7.We corroborated that our results related to the band weight were according to Cha et al., (2019), Jeong et al., (2023), and Seijsing et al., (2018). For our case, just 6 hours were required for the protein expression.

Figure 9. LysCSA13-ABD 0.2 mM IPTG protein induction kinetic. A) ColorBurst protein ladder. B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysCSA13-ABD non-inducted, fourth hour. D) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, fourth hour. E) E. coli BL21 (DE3)- LysCSA13-ABD non-inducted, fifth hour. F) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, fifth hour. G) E. coli BL21 (DE3)- LysCSA13-ABD non-inducted, sixth hour. H) E. coli BL21 (DE3)- LysCSA13-ABD inducted at 0.2 mM IPTG, sixth hour. I) E. coli BL21 (DE3), first hour.

Figure 10. LysK-ABD-SH3B30 0.2 mM IPTG protein induction kinetic. A) ColorBurst protein ladder. B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysK-ABD-SH3B30 non-inducted, fourth hour. D) E. coli BL21 (DE3)- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, fourth hour. E) E. coli BL21 (DE3)- LysK-ABD-SH3B30 non-inducted, fifth hour. F) E. coli BL21 (DE3)- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, fifth hour. G) E. coli BL21 (DE3)- LysK-ABD-SH3B30 non-inducted, sixth hour. H) E. coli BL21 (DE3)- LysK-ABD-SH3B30 inducted at 0.2 mM IPTG, sixth hour. I) E. coli BL21 (DE3), first hour.

Figure 11. PCNP-CecA-LysSS expression. Visualization through a polyacrilamide gel 15% (SDS-PAGE) results. A) MWM (iBright). B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- PCNP-CecA-LysSS non-inducted. D) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 0.2 mM IPTG. E) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 0.5 mM IPTG. F) E. coli BL21 (DE3)- PCNP-CecA-LysSS inducted at 1.0 mM IPTG

Insouble and soluble protein fractions

Once the best protein induction parameters were established, a new SDS-PAGE was performed to assess the protein expression site in the cell. Our endolysins were induced at 0.2 mM IPTG, for 5 (LysK-ABD-SH3B30) and 6 (LysCSA13-ABD) 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 LysCSA13-ABD an insoluble fraction has been demonstrated (Figure 12). On the other hand, LysK-ABD-SH3B30 yielded typical results of a soluble protein (Figure 13).

Figure 12. LysCSA13-ABD (A) soluble and (B) insoluble fractions. A) Both gels presented the same distribution of wells, each one corresponding to the fraction (soluble or insoluble) assigned. A) MWM (for gel A: Precision Plus Protein Dual Color Standards (10–250 kD), for gel B: PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa). B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysCSA13-ABD non-induced. D) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 2 μL. E) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 4 μL. F) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 6 μL. G) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 8 μL. H) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 10 μL. I) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 15 μL. J) E. coli BL21 (DE3)- LysCSA13-ABD induced at 0.2 mM IPTG, 20 μL.

Figure 13. LysK-ABD-SH3B30 (A) soluble and (B) insoluble fractions. Both gels presented the same distribution of wells, each one corresponding to the fraction (soluble or insoluble) assigned. A) MWM (for gel A: Precision Plus Protein Dual Color Standards (10–250 kD), for gel B: PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa). B) E. coli BL21 (DE3). C) E. coli BL21 (DE3)- LysK-ABD-SH3B30 non-induced. D) E. coli BL21 (DE3)- LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 2 μL. E) E. coli BL21 (DE3)- LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 4 μL. F) E. coli BL21 (DE3)-LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 6 μL. G) E. coli BL21 (DE3)- LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 8 μL. H) E. coli BL21 (DE3)- LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 10 μL. I) E. coli BL21 (DE3)- LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 15 μL. J) E. coli BL21 (DE3)-LysK-ABD-SH3B30 induced at 0.2 mM IPTG, 20 μL.

Third Round: Protein Purification

The current section emphasizes on the obtaining of the specific fusion-endolysins, separating them from the crude protein extracts. This is possible because of the previous in silico design, in which we incorporated a 6X HisTag to its purification by using a metal ion affinity chromatography column.

To perform the protein purification, we followed the protocol described in the Experiments Page. For the case of the LysK protein, we correctly identified the purified protein through a 12% polyacrylamide electrophoresis SDS-PAGE by using our soluble fraction collected from the crude protein, which was inducted at 0.2 mM IPTG at the optimal conditions that are described in the previous sections of this page. The protein was identified as is shown in Figure 14.

Figure 14. LysK purification. A) PageRuler™ Plus Prestained Protein Ladder. B) E. coli BL21 (DE3). C) E. coli BL21 (DE3) LysK- non-inducted. D) Wash buffer 1. E) Wash buffer 2. F) Wash buffer 3. G) Elution buffer 1. H) Elution buffer 2. I) Elution buffer 3.

Fourth Round: Bactericidal Assays

For this stage the main purpose was to corroborate the catalytic activity of our endolysins, the optimal parameters, and the real effects for each bacteria. In the same way, the chitosan-alginate delivery drug system was analyzed simultaneously with our enzymatic treatment.

Considering our protein purification results, we decided to work on the development of our treatment by using our crude protein extracts. To identify our optimal composition, we designed an experimental model that combines simultaneous enzyme analysis and its effects on the pathogenic bacteria. The full protocol is described on our Experiments Page. Our ELISA plate design was prepared as follows:

Figure 1. Distribution of ELISA plates for bactericidal assays.

Figure 2. Distribution of ELISA plates for bactericidal assays with loaded protein chitosan alginate beads.

Figure 3. Evaluation of the chitosan-alginate beads in comparision with the raw protein extract mix, S. aureus.

Figure 4. Evaluation of the chitosan-alginate beads in comparision with the raw protein extract mix, against S. agalactie.

Figure 5. Evaluation of the chitosan-alginate beads in comparision with the raw protein extract mix, against E. coli.