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Design

Strategic Design

Our objective is to engineer a living microbial system for use in foot care, embedded in a nutritive foot-bed. This innovative approach aims to solve common foot care issues that people experience daily, such as fungal infections, inflammation, and odor. By leveraging the ability of genetically engineered bacteria to continuously produce a beneficial compound, we aim to create a solution that goes beyond traditional creams and ointments.

Specifically, we aim to use bacteria that produce linalool, our target compound, a naturally occurring molecule found in plant-based oils. Linalool offers multiple properties that align with foot-care: it is anti-fungal, anti- inflammatory, fragrant, and non-toxic. The living microbial system will be embedded within a nutritive foot-bed, allowing the genetically modified bacterium to use the substrate to sustain itself, produce and release linalool, which will diffuse throughout the foot environment.

Live Application of Engineered Bacteria

In contrast to conventional treatments that require frequent application, lower regions of applicability and have a limited active window, our approach integrates a living microbial system into a nutritive foot-bed, turning it into a long-term care solution. This foot bed is designed to fit into any shoe, where the bacteria embedded in the foot-bed use the nutrients within the foot-bed substrate to synthesize and secrete linalool over an extended period.

Why Use a Living Microbial System in the first place?

The advantage of using a living microbial system is that it provides continuous, self-sustaining production of therapeutic molecules. By housing bacteria in a contained and controlled environment within the foot-bed, the system can deliver round-the-clock foot care. Instead of relying on externally applied products that wear off after a few hours, the living microbial system ensures that fresh linalool is produced as long as the foot-bed is in use, forming an intrinsic part of the foot environment with time.

Linalool: The Perfect Foot Care Molecule

Linalool is a naturally occurring terpene alcohol found in many plants, such as lavender and coriander.It was specifically chosen for this project because of its multifaceted properties:

  • Anti-inflammatory and Antifungal Properties: Linalool has been shown to reduce inflammation, making it particularly effective in soothing irritated skin. Its antifungal activity can help manage common foot-related conditions, such as Athlete’s Foot.
  • Fragrance and Non-toxicity: As a pleasant-smelling and non-toxic compound, linalool adds a fragrance to the foot-bed, improving the user experience and combating bad odors. The compound is generally regarded as safe for topical use, even over long periods.
  • Easy Diffusion: One of the key benefits of linalool is its volatile nature, allowing it to escape easily from the nutritive foot-bed and disperse throughout the shoe. This diffusion ensures that the entire foot is exposed to the compound, providing continuous protection and skincare even in hard-to-reach areas.
Population with QS

E. Solei: Product Envisioning - Lifting the Veil!

As the user walks, the bacteria embedded in the nutritive foot-bed metabolize the substrate and convert it into linalool, which then diffuses into the surrounding environment (the shoe and foot). This continuous release ensures that the foot is always in contact with linalool, providing round-the- clock protection against fungal growth, inflammation, and bad odor.

By creating a living ecosystem, this foot-bed offers a longer-lasting solution compared to topical applications, which are either washed off or worn away. Importantly, this system requires minimal user intervention, making it a hassle-free foot care solution.

Choosing the Chassis: Why Lactobacillus rhamnosus?

Why is Lactobacillus rhamnosus an ideal candidate for the foot-bed application for our iGEM project?

Safety Diagram
  • Safety and Probiotic Nature: Lactobacillus rhamnosus is a well-established probiotic, commonly found in the gut and known for its safe use in various health applications. It has a proven track record in human-related treatments without causing harm, even when used in delicate environments like the gastrointestinal and urogenital tracts. This makes it a suitable candidate for integration into a nutritive foot-bed, minimizing the risk of harm if it comes into contact with skin or accidental exposure.
  • Antifungal and Antimicrobial Properties: Lactobacillus rhamnosus is known for producing antimicrobial compounds like 3-phenyllacticacid, which exhibit strong antifungal properties. This aligns with the production of linalool, a compound with antifungal and anti-inflammatory properties, thereby complementing it and enhancing the foot-bed's efficacy in managing skin health and preventing fungal infections (Athlete's foot, etc.).
  • Robustness and Skin Microbiome Compatibility: This species of Lactobacillus is resilient in various conditions, including acidic environments and bile salts, which makes it adaptable for the relatively dynamic environment of a foot-bed. It can also support the skin’s natural micro-biome by outcompeting harmful pathogens without disrupting the overall microbial balance, which is critical in preventing skin irritation or infections.
  • Immunomodulatory Potential: Lactobacillus rhamnosus has been shown to have immunomodulatory effects, potentially aiding in reducing inflammation and improving skin health. Its presence can help maintain a healthy immune response on the skin, adding an additional layer of protection against pathogens.
  • Thus we take home the fact that Lactobacillus rhamnosus provides a package of safe antimicrobial action, and skin- compatible properties, making it an ideal candidate for use in a live microbial system in the foot-bed designed to release therapeutic molecules such as linalool and manage skin conditions.

    Further details can be extracted from discussions on Microbial Cell Factories and Applied Microbiology and Biotechnology.

    For our project, the team carries out all experiments in Escherichia coli (BL21 and DH5- alpha strains) as chassis, which are to be replicated when the chassis is changed to Lactobacillus rhamnosus.

Safety Considerations for Living Microbial Systems in Footbeds

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  • Use of Non-Pathogenic Strains: The bacteria selected, Lactobacillus rhamnosus, is a safe probiotic strain that is non-pathogenic and commonly found in the human microbiome. It minimizes health risks while performing the desired function in the footbed.
  • Physical Barrier Between Footbed and Environment: The footbed is designed with a physical barrier separating it from the external environment. This barrier ensures that bacteria remain contained within the footbed and prevents any unintended release of microbes into the surroundings, providing an additional layer of safety.
  • Auxotrophy for Safety: The bacteria are engineered to be auxotrophic, making them dependent on specific nutrients provided by the footbed media. This ensures that the bacteria cannot survive or proliferate outside the controlled environment of the footbed, reducing the risk of environmental contamination.
  • Minimalist Cell Design: The bacterial cells are genetically streamlined, only containing the essential components necessary for producing beneficial compounds like linalool. This reduces the risk of unforeseen genetic interactions or behaviors.
  • Kill Switch: The living microbial system incorporates a kill switch, ensuring the bacteria can be safely deactivated or killed under specific conditions, such as environmental exposure or the end of the product’s lifespan, or detachment from the media layer.
  • Safe Disposal Methods: Disposal protocols are put in place to neutralize any remaining bacterial activity and prevent live bacteria from escaping into the environment after the footbed has been used.
  • Controlled Population Growth: The bacterial population within the footbed is regulated through a quorum sensing mechanism, controlling cell division and preventing overpopulation. This control minimizes the risks of nutrient depletion, toxic metabolite buildup, and overgrowth.
  • Dependence on Footbed Media: Due to their auxotrophic nature, the bacteria are reliant on the nutritive footbed media for survival, which means they cannot propagate outside the footbed. This makes the microbial system self-limiting and enhances its biosafety.

These multi-layered safety measures ensure that the microbial system is both effective and secure for long-term use in footbed applications, while protecting both the user and the environment.

Addressing a major problem with living bio-material therapeutics

When integrating living microbial systems into applications such as a nutritive footbed, there are several inherent issues related to population control and environmental sustainability. Without proper mechanisms to manage bacterial growth, these systems can quickly become inefficient due to several factors:

  • Uncontrolled Population Growth: Bacteria in a nutrient-rich medium can rapidly proliferate, leading to overpopulation. This unchecked growth overwhelms the medium’s capacity, causing rapid nutrient depletion.
  • Nutrient Depletion: As bacterial numbers increase, they consume the available resources within the medium faster than it can be replenished. This results in a shorter functional lifespan for the microbial system.
  • Toxic Metabolite Buildup: Excessive bacterial growth leads to the accumulation of metabolic byproducts. Without regulation, these byproducts can become toxic to the bacteria themselves, ultimately reducing the system's overall efficacy.
  • Loss of Stability: Overcrowded bacterial populations experience stress from competition for limited resources. This stress can lead to decreased productivity and failure to produce the desired compounds, such as linalool in the footbed application.

    produce the desired compounds, such as linalool in the footbed application. These challenges highlight the key limitations of using living microbial systems in practical, long-term applications.

Facing the Challenges

Our solution to the bacterial population problem in the footbed involves a dynamic system that intelligently automates itself and controls bacterial growth to maintain a stable and healthy population. This system ensures the bacteria never overgrow, which could lead to the accumulation of harmful byproducts, or die off too quickly, which would reduce their beneficial effects. By periodically pausing their ability to multiply, the system prevents the bacteria from exceeding the capacity of their environment. This controlled population management extends the lifespan of the footbed by preserving the nutrients in the media and allowing the bacteria to continuously produce beneficial compounds, like linalool, over an extended period. This way, we create a sustainable, safe, and efficient solution that supports long-term foot care without the need for frequent replacements or interventions.

Traffic Police Analogy

The system works much like traffic police, regulating bacterial "traffic" to ensure smooth operation. The bacteria are allowed to multiply when needed, but once the population reaches optimal levels, the system "halts" further growth until necessary. This way, it balances population stability and efficiency.

Phases of Development

  • Phase 1 - Design and Proof of Concept:
    • Design & incorporate population regulation model.
    • Characterize population regulation model.
    • Incorporate & characterize linalool-producing circuit.
    • Develop initial kill switch mechanism.
    • Test bacteria for proof of concept in lab.
    • Initial testing of media and viability.
    • Refine based on initial results.
  • Phase 2 - System Integration:
    • Finalize & incorporate kill switch.
    • Co-transform population & linalool circuits.
    • Synthesize & optimize media for bacterial growth.
    • Test media-bacteria compatibility.
    • Small-scale tests for population control & linalool production.
  • Phase 3 - Live Testing:
    • Conduct live testing in real-world conditions.
    • Measure population stability & linalool production.
    • Test kill switch & safety mechanisms.
    • Refine system based on test data.