Implementation

Production

Lab-Level Production Strategies (as implemented by the team in the course of our experiments on E. coli. Similar procedures are supposed to come into play on switching our chassis to Lactobacillus rhamnosus).

Preparation of Competent Cells: Using established techniques, competent E. coli cells are prepared as the first step. We have employed the TFB1, TFB2, and CaCl2-RbCl technique. The electroporation technique might improve the cells' capacity to absorb exogenous plasmid DNA, which is essential for a satisfactory transformation.
Cotransformation:After competent cells are prepared, two different plasmids are to be concurrently injected into the competent cells using a cotransformation technique. To increase the absorption efficiency, the plasmid concentrations and ratios must be optimised.
Selection and Screening: After transformation, cells are plated on media that are specifically designed to withstand the antibiotics that match the resistance genes expressed in the plasmids. Effective colonies are selected using molecular methods.
Small-Scale Cultivation: Positive colonies are to be inoculated into small liquid cultures (e.g., LB broth supplemented with antibiotics) to facilitate further growth and analysis of the transformed E. coli strains.

Bulk-Level Production Strategies with Lactobacillus rhamnosus

Scale-Up of Competent Cells: For bulk production, large-scale cultures of Lactobacillus rhamnosus are to be grown to high cell densities prior to competency induction. Fermentation techniques may be employed to optimise growth conditions, including control over nutrient availability, pH (very slightly acidic conditions optimises growth), and aeration.
Co-transformation in Bioreactors: The co-transformation process can be conducted in bioreactors, allowing for greater control over environmental parameters. Optimization of mixing and oxygenation is crucial to ensure effective plasmid uptake by the competent cells.
High-Throughput Screening: Implementing high-throughput screening methodologies enables rapid identification and isolation of transformed colonies. Automated systems for colony picking and subsequent PCR validation can significantly enhance efficiency and throughput.
Cryopreservation: Upon successful transformation and growth, transformed Lactobacillus rhamnosus strains can be subjected to cryopreservation techniques to ensure long-term viability and plasmid stability. This process facilitates the preservation of the genetic constructs for future applications.
Scale-Up Culture Conditions: Optimising culture media (without peptone but containing glucose and skim milk powder) and growth conditions (e.g., temperature, slightly acidic pH, agitation) within larger fermenters is essential for maximising the yield of the transformed Lactobacillus rhamnosus. Continuous monitoring of growth parameters is recommended to maintain optimal conditions. Also new techniques like semi-solid fermentation (SSF) can be deployed to achieve high cell density populations of the chassis.
Quality Control: Comprehensive quality control measures should be implemented at various stages of production, including assessments of plasmid stability and gene expression levels. Techniques such as restriction enzyme analysis and sequencing may be employed to verify the integrity of the transformed constructs.

Production References

Sambrook, J., & Russell, D. W. (2001). "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory Press.
Inoue, H., Nojima, H., & Okayama, H. (1990). "High efficiency transformation of Escherichia coli with plasmids." Gene, 96(1), 23-28. doi:10.1016/0378-1119(90)90261-I.
Gibson, D. G., et al. (2009). "Enzymatic assembly of DNA molecules up to 1 Mb in size." Nature Methods, 6(5), 343-345. doi:10.1038/nmeth.1318.
Zhang, Y., & Wang, Z. (2018). "Advances in microbial cell factories for the production of biochemicals." Biotechnology Advances, 36(5), 1255-1267. doi:10.1016/j.biotechadv.2018.01.002.
Khan, A. M., et al. (2018). "High-throughput transformation of Escherichia coli for plasmid production." PLoS ONE, 13(10), e0205722. doi:10.1371/journal.pone.0205722.
Huang, Y., et al. (2020). "Cryopreservation of bacteria: a review of current strategies." Microbiology, 166(1), 1-9. doi:10.1099/mic.0.000885.

Packing

Choice of Polymer

You look around right now, sir and almost everything you set eyes on or touch, from your toothbrush bristles, to the pen you are holding, is made from a polymer. A few words on polymers? They are large compounds made of repeating units called monomers. Some naturally-occurring polymers include cellulose, natural rubber, etc. In this world of dynamically changing industries polymer forms the basis of many a one, including textiles, food, etc. and it forms an integral part of our project E.solei as well.

Our GMO is designed to sustain itself in a bio-compatible polymer, which would contain the nutrients necessary to promote normal physiological growth of the GMO and subsequently linalool production.

We propose an alginate-collagen brush polymer for housing our GMO, a Lactobacillus rhamnosus species, which has excellent binding properties to the surface of polymers, because of its distinguished hydrophobicity. The biopolymer, during its manufacture, is infused with nutrients required to keep the bacteria growing and producing the compound Linalool. Of course, the choice of polymer is kept with cost in mind, with it being one of the cheapest available materials.

Delving Deep

Composition

Alginate and its Suitable Properties

Alginate is a hydrocolloid from algae, specifically brown algae, which is a group that includes many of the seaweeds, like kelps and an extracellular polymer of some bacteria. Sodium alginate is one of the best-known members of the hydrogel group.

Alginate is biocompatible, biodegradable and non-toxic, has high biosafety and undergoes rapid ionic gelation, is nutritious to bacteria and therefore makes up an ideal composition for the matrix.

Collagen, Chitosan and their Roles

Ionically cross-linked alginate hydrogels have the disadvantages of insufficient mechanical properties and long-term stability owing to ion-exchange.

Here collagen and other stable materials such as chitosan come into play. Collagen, the most abundant protein in our body, can be used in combination with alginate in the form of a hydrogel. This collagen-alginate polymer increases the mechanical stability and use of fish collagen also further reduces swelling.

Alginate also shows pH sensitivity and might degrade at very low pH("<4>") Even though our foot environment seldom registers pH levels below 4, we can increase the robustness of alginate towards a low pH condition, by mixing chitosan with it

STRUCTURAL ATTRIBUTES

Use of Brush Polymers

A polymer brush is the name given to a surface coating consisting of polymers tethered to a surface. These polymer layers can be tethered to flat substrates such as silicon wafers, or highly curved substrates such as nano-particles. Also, polymers can be tethered in high density to another single polymer chain, although this arrangement is normally named a bottle brush.

The brushes are often characterised by the high density of grafted chains. The limited space then leads to a strong extension of the chains. Grafting polymers to a surface to form a polymer brush is often performed to create coatings, as polymer brushes favourably modulate bacteria.

We propose to use a similar brush polymer made from alginate-collagen composition, to whose brush-like extensions our GMO will adhere due to its surface hydrophobicity.

Adherence of GMO

Our GMO, Lactobacillus rhamnosus, has high hydrophobicity due to the composition of its cell surface, which includes the occurrence of hydrophobic amino acids, polysaccharides. Due to increased hydrophobicity it can easily adhere to the bristles of the brush polymer.

State of GMO: The GMO is lyophilised and then attached to the surface of the bristles. This is primarily done to ensure dormancy till the point the user opens the encasing and uses the product. This causes no wastage of either product or resources (both bacterial and nutrient).

How Will Our Product Look Like? : Packaging and Usage

The product comes packaged in two layers: Layer 1 and Layer 2, with a similar brush polymer and lyophilised bacteria adhered to the surface. Both the layers come packaged in a porous casing, with a pore size such that it does not let the GMO to pass through but allows free diffusion of our target molecule Linalool.

Before the usage, the encasing is removed from top. An inducer medium is sprayed on top of the layers. This revives the bacterium from its lyophilised state so that it carries on a normal life cycle henceforth. Now layer 1 is inverted and placed face down on layer 2 and both the layers are covered by the encasing. This complete bi-layer is now placed inside the shoe sole.

Usage

Packing and Choice of Polymer References

Disposal

Disposal of Product

It is important to ensure proper disposal mechanisms for the Genetically Modified Organism (GMO) proposed to release in an environment outside laboratory conditions. Improper disposal of engineered organisms can cause different dreadful mutations, including horizontal gene transfer, cancer, and severe allergies, leading to harmful effects on humans as well as other animals. Environmental hazardous chemicals can be released by mutated engineered organisms in case of faulty disposal measures.

However, our proposed product, E. solei, will ensure safety through disposal strategies for every layer of manufacturing.

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Strategy for Hosting Bed

Perspective 1: Alginate

As suggested in the packing section, the hosting material of the product will be composed of Alginate, a hydrocolloid obtained majorly from brown algae. It is non-toxic to tissues and cells, hence no possible harm by skin contact on accidental exposure to the broken product cassette. Alginate can be degraded by exposure to high temperature (above 250°C), and the alginate matrix can be dismantled by cation chelation. Recyclability is ensured by the pH control of the chelator.

Perspective 2: Collagen and Chitosan

Collagen, being a major structural component of most animals, is completely biodegradable. Collagen is made with three different polypeptide chains forming a particular structure to perform certain physiological activities. Hence, collagen is degraded by enzymatic degradation. It has no harmful effect on exposure to animals or humans.

Chitosan, on the other hand, is a biopolymer profoundly used for its biodegradability and low toxicity. It is also degraded by lysozyme enzyme exposure. Research shows that chitosan is degraded into non-toxic residues more quickly in the soil. Additionally, it is not harmful to animals or humans.

Strategy for Structure and GMO

Perspective 3: Brush Polymers

Brush polymers are composed of several polymer chains in a specific organization to perform the desired function. These are recycled by exposure to high temperature, degraded by the action of microorganisms, and are eco-friendly, which is why they have diverse biomedical applications.

Perspective 4: Lactobacillus rhamnosus

The large group of Lactobacillus is considered harmless to humans as well as animals. Moreover, Lactobacillus rhamnosus is a probiotic bacteria. A probiotic bacteria is safe for even human consumption in forms of drugs, so there will be no harm to the environment in case of accidental release of the GMO.

Besides, our proposed kill-switch machinery adds extra safety to the user by turning on the automated killing of the bacteria upon detachment from its substrate. Moreover, as our GMO comes in a lyophilized form, it is inactive until the activation of the product by spraying media onto it. Hence, any unintentional release or breakage of the product before use will not cause any harm to the surroundings or users. In its lyophilized form, the bacteria can be stored for a long time.

Disposal Collection and Safety

As every layer of our product is biodegradable or recyclable with no harm to the environment, the material will be collected in a disposal bag provided with the product packaging, labeled as "recyclable", for sorting collection by the waste management personnel in the area.