Proof of concept

Let creativity dream beyond limits.

Overview

As the iGEM Tec-Chihuahua 2023 team, we have identified a critical issue in our state of Chihuahua, Mexico – the presence of mastitis disease, which significantly impacts the dairy industry, a cornerstone of the state's economy. Currently, the primary treatments involve antibiotics, which pose a health risk to consumers as these can pass into the milk produced by cows undergoing mastitis treatment. Our product, AureoBos, offers a promising solution. By utilizing novel fusion proteins combining endolysins, albumin binding domains, and cell wall binding domains, we aim to create a highly effective antimicrobial agent. Fusion proteins, a cutting-edge technology, hold immense potential in biopharmaceuticals, allowing proteins to target specific molecules or cells. Our product not only seeks to outperform current treatments but also addresses the challenges associated with antibiotic use in bovine mastitis treatment.

Impacting the world by using synthetic biology is the core of Aurebos, thanks to the development of biological devices, we found and solved a huge socio-economic problematic which directly impacts into our city, Chihuahua. But it was not always the same, not long ago, we were only a pretty schoolar project which only take care of the science reported on papers. At the very begining, AureoBos was composed by two endolysins which would were administrated by the intravenose way at the infected cow mammary glands, targeting S. aureus which was considerated as the main bacteria ifection precursor. But our product started to grow when AureoBos interacted with the real world through it's stakeholders suggestions.

Integrated Human Practices

In fact, AureBos would be completely different without the stakeholder suggestions, from the composition of the bactericidal treatment up to the administration method. Some of the most important uptakes that make AureoBos the inframammary infusion treatment that is is going to revoluzionate the dairy industry are described as follows:

Application method

Our application method changed during the development of our project, as we gained different insights from our stakeholders. We decided to change it from a topical ointment to an intramammary infusion as they told us it would have a better performance. To know more about the reflection, visit out Human Practices Page

New microorganism

After our research we discovered that mastitis is caused by three main microorganisms, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis. The more we delved into the problem, we realized that beyond theory, in the field E.coli possesses a significant challenge for the dairy industry. This realization marked the initiation of our research into a novel endolysin capable of combating E. coli.

Chitosan & Alginate

In our quest to enhance our delivery system, we embarked on a comprehensive examination of the competitive landscape. As we delved into our research, a noteworthy observation emerged: all existing formulations relied on transportation vehicles.

Considering that our product was intended for intramammary use, we chose to explore formulations commonly employed in intravaginal products designed for humans, as our previous attempts using animal formulations had yielded no success.

During our investigation into suitable carriers for our endolysins, we uncovered findings that pointed to the effectiveness of alginate-chitosan nanoparticles as a promising vehicle, experts in the pharmaceutical field have voiced their agreement, highlighting that nanoparticles serve as excellent drug carriers. Notably, chitosan possesses antimicrobial properties, a feature that could potentially booster the effectiveness of AureoBos.

AureoBos as a Startup

Our business model has been meticulously designed and rigorously validated through an extensive engagement with a diverse number of experts, both within and beyond the entrepreneurial ecosystem of our university, such as Zoetis, MSD Animal Health, Ilum Venture Capital, StartUp Mexico, among others. Through active participation in local and national forums, we have garnered validation from industry experts and the wider startup community, conclusively establishing AureoBos as a scalable and viable startup. For a deeper dive into the specifications of our business model, strategic approach and experts' feedback, we invite you to explore the entrepreneurship section.

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.

Bactericidal Assays Results

Through the bactericidal assays, it was possible to identify the experimental effect of our fusion endolysin treatment combination against S. aureus, S. agalactie, and the E. coli pathogenic bacteria.

The design of our process was deeply described in the Experiments Page (Figure 1). Our results corroborate the lysing efficiency of our treatment against the mastitis causing bacteria.

To determine the percentage of bactericidal effect from each raw protein extract, the following equation was developed: (100- (sample fluorescence value*100)/(fluorescence from the positive control-fluorescence from the negative control).

Figure 1. Bactericidal assay plate distribution.

In the same way, we are assured of the potentialization of the bactericidal effect of our treatment by the incorporation of chitosan-alginate vehicles, in which:

Figure 2. Evaluation of the chitosan-alginate beads in comparision with the raw protein extract mix, S. aureus.

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

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

As can be appreciated in Figures 2 to 4, the addition of the chitosan-alginate beads increased the bactericidal activity for each bacterium, a fact that couldn't be completely developed without the stakeholder's suggestions.

Math Model

Response Surface Methodology (RSM) is a surrogate modelling technique that combines mathematical and statistical techniques valuable for construction models and analyzing scenarios where multiple independent variables or controllable factors influence a dependent variable or response. RSM efficiently extracts a wealth of information through minimal effort.

Response Surface Model

For the model section, an analysis of a three-level full factorial response surface model for the investigation of the variation of each of our fusion-proteins and its relation to the pathogenic bacteria was the main purpose.

Figure 6. Response surface graph of coded values, from -1 to 1, representing the low and high limits of protein concentration for LysK and LysSS, which are 875-2625 μg/mL and 25-75 μg/mL, respectively. The response is quantified in terms of the percentage of S. agalactie killed.

The quadratic model for bactericidal activity in S. aureus after treatment is significant, with LysK and LysCSA13 being significant factors affecting bactericidal activity. Both LysK and LysCSA13 contribute positively to bactericidal activity, with their quadratic effect being particularly influential for LysCSA13.

The 2FI model for bactericidal activity is significant, with LysK (A), LysSS (C), and the interaction term AC being significant factors affecting bactericidal activity. Interestingly, the model establishes that interaction between LysK and LysSS has a synergistic effect on bactericidal activity. This model aligns with reality, considering that LysK exhibits a broad spectrum of target bacteria, with S. agalactiae being one of them. The potential synergistic effect observed with LysSS could be attributed to its low protein concentration, suggesting that it might contribute to the overall effect observed with LysK. Moreover, the interaction between LysK and LysSS has a notable negative impact, resulting in a significant decrease in viable cells when both factors are present simultaneously.

Figure 7. Response surface graph of coded values, from -1 to 1, representing the low and high limits of protein concentration for LysK and LysCSA13, which are 875-2625 μg/mL and 500-1500 μg/mL, respectively. LysSS is coded at -1 representing 25 μg/mL. The response is quantified in terms of the percentage of E. coli killed.

ANOVA analysis indicated the lack of statistical significance for the terms associated with LysSS (C values), the graphical representations above clearly illustrate subtle variations in the response when altering the C value, represented by coded values ranging from -1 to 1 (25 to 75 μg/mL). In the biological and experimental context, it is possible to assume that, as a consequence of the complex process of synthesizing the PCNP-CecA-LysSS protein in our cell host (because of the cell's toxicity against E. coli), the final protein concentration was very low, enough to dilute the bactericidal effect to the point of being statistically insignificant.

Through our Math Model was possible to confirm, predict, and simulate the bactericidal assays in order to optimize the experimental designs. Considering all the background developed on our Math Model page, AureoBos incorporates all the stakeholder suggestions, and the optimal parameters for each stage of the synthesis process are estimated.

Entrepreneurship

AureoBos is set to revolutionize the dairy industry with its pioneering intramammary infusion treatment. By offering dairy producers an innovative solution for addressing bovine mastitis, caused by the four most common microorganisms Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, and Escherichia coli we eliminate the reliance on antibiotics.

Bussines model

Through our human practices, we successfully pinpointed the genuine needs of our end-users. Recognizing the significant shortcomings of existing solutions, we identified the key areas of need and made the decision to develop AureoBos: an intramammary infusion free from antibiotics. Our aim is not only to reduce costs and enhance food safety but to completely revolutionize the treatment of bovine mastitis.

Building upon this insight, we took the initiative to structure our entire business model. In doing so, we identified two primary customer segments: large-scale dairy producers and veterinary pharmaceutical companies that will serve as a channel to reach small and medium-sized producers.

Simultaneously, we charted the course for the future to bring AureoBos to life. We identified the critical activities, essential resources, and key partners needed for this endeavor. We also conducted a thorough legal analysis to ensure the formal establishment of the startup while safeguarding its scalability.

To validate our progress as a startup, we actively participated in various pitch events, seeking feedback from industry experts and the entrepreneurial community. Our dedication paid off as we secured the top position in the national category of validated ideas at the UP Innovation Awards 2023, thanks to it, AureoBos obtain a prize of USD 5,627$ for the entrepeneurship development. All of these efforts have allowed us to establish a solid foundation for validating and expanding our startup.

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