Introduction

Adhesives are used in everything from household repairs to industrial manufacturing. The industry is growing rapidly, with sales increasing by one billion USD annually [1]. Recent research on adhesive materials focuses on natural adhesive proteins secreted by barnacles [2]. However, bacterial adhesive polysaccharides, known as holdfasts, exhibit distinct characteristics and possess tremendous potential for development. These polysaccharides feature ultra-high viscosity, which can be leveraged to design devices with strong adhesion requirements. 

High biocompatibility of bacterial adhesives presents promising opportunities in the medical field, particularly for implantable medical devices: EKG sensor patches, transcutaneous electrical nerve stimulation (TENS) patches for chronic pain, negative pressure wound therapy (NPWT), and hormonal therapy. Medical patches, while convenient, still cause allergic reactions or skin irritations [9][10]. Diabetes affects 537 million people worldwide and causes 1.5 million deaths annually, a number that is expected to increase. Specialized adhesive patches holding insulin pumps and glucose sensors peel off prematurely and cause skin allergies. These problems are dangerous and expensive, as these devices cost up to $5,000 [3][4].

Synhesion aims to create natural bacterial glue and offer a patch-sized solution for millions. We harness adhesive-producing biochemical pathways from aquatic bacteria and transfer them to industrially used E.coli bacteria for about 7 times faster and more controllable growth. Glue can be synthesized using glucose - a cost-effective, renewable feedstock. In contrast, the strongest available glue, epoxy resin, requires expensive oil-based chemical precursors. By doing this, we not only make our production process suitable for industrial use but also develop a method to create superglue that doesn't rely on non-renewable oil-based materials. Our method uses sustainable, low-cost glucose instead.

The holy grail of glue

Caulobacter crescentus is a freshwater, gram-negative bacterium known for its unique dual lifestyle. It transitions from a motile "swarmer" cell with a flagellum for chemotaxis to a "stalked" cell, which adheres to surfaces with an adhesive called holdfast. Only a few studies in recent years explored the holdfast structure, and none tried to recreate the synthesis pathway in E. coli. Preliminary studies on the physicochemical properties of holdfast have revealed that the C. crescentus holdfast is the strongest discovered bioadhesive [5][6][7][8].

Adhesive-producing biochemical pathway

Plasmid construction

To address the challenge of transferring 12 genes, we divided them into two groups: sugar tetrad assembly proteins (hfsEJGLHK) and polymerization-export proteins (hfsABDFCI). These genes were distributed across two plasmids, each regulated by two T7/lac promoters for efficient expression. We chose two compatible vectors for gene cloning: the pRSFDuet vector for sugar tetrad assembly proteins and the pACYCDuet vector for polymerization-export proteins. The low-copy pACYCDuet vector minimized the formation of inclusion bodies, while the high-copy pRSFDuet vector ensured sufficient protein concentration for tetrasaccharide assembly. We transferred the adhesive synthesis pathway to industrially used E.coli bacteria for about 7 times faster, cheaper, and more controllable growth.

Adhesive biosynthesis

Since no one has done it before, we took our time to optimize the polysaccharide adhesive production pathways in E. coli. By varying expression and growth temperatures and IPTG concentrations we found an optimal combination for holdfast synthesis (see Results). On top of that, multi-sugar polymer biosynthesis requires specific materials to be functional, so choosing an appropriate substrate is also crucial. We tried out 8 different substrates for holdfast synthesis and, using various verification methods and modeling, chose glucose as an optimal one.

Making adhesive production efficient

We aimed to enhance the efficiency of polysaccharide adhesive production by reducing costs and minimizing waste. To achieve this, we focused on lowering the metabolic stress on E. coli and selected a similar polysaccharide-producing ECA pathway as our target to eliminate competition for shared substrates, ensuring a more efficient and cost-effective adhesive production process.

The ECA polysaccharide synthesis pathway in E. coli is analogous to the holdfast in C. crescentus. Both pathways involve the assembly of polysaccharides, complex molecules composed of multiple sugar units.

Finally, by using the Red/ET homology recombination system, we removed the wecA gene and disrupted the ECA pathway in E. coli protein expression strains (see Results). We reduced the metabolic stress from E. coli while increasing substrate availability for holdfast biosynthesis.

Producing the glue

After polysaccharide synthesis in E.coli, the adhesives were purified from the media for further investigation or usage.

Only glue?

Individual proteins from the holdfast synthesis pathway can be used for a completely different yet important purpose. Tetrad assembly proteins (HfsE, HfsG, HfsH, HfsJ, HfsK, HfsL) build a short chain of sugar monomers in a specific sequence, creating a glycolipid—a phospholipid with a covalently attached carbohydrate molecule.

Glycolipids are primarily located on the extracellular surface of eukaryotic cell membranes and are responsible for various functions, such as acting as receptors for viruses and other pathogens. These receptors allow pathogens to enter specific host cells with unique glycolipid markers. This feature could enable the use of glycolipids as labels for precise and targeted liposome distribution throughout the body, delivering anything from cancer drugs to gene-editing systems directly to target cells.

Establishing expression and purification strategies for these specific proteins provides a strong foundation for the potential use of glycolipids as labels for targeted drug delivery, offering future iGEM teams innovative ideas for liposome-based therapies.