In the world of waterproof adhesives, we face a pressing issue: the environmental impact of synthetic adhesives, particularly those containing PFAS. These chemicals are durable but hazardous, posing risks to both the environment and human health. While efforts are underway to regulate their use, out iGEM team is exploring an exciting alternative. Caddisfly silk, produced by aquatic caddisfly larvae living in water, has remarkable adhesive properties. It's resistant to water, heat, and non-toxic to human cells. What sets it apart is its natural stickiness, making it an ideal underwater adhesive. We're looking to produce caddisfly silk through synthetic biology in bacteria, providing a more sustainable and scalable method. This natural adhesive could offer a valuable solution, especially when we consider the drawbacks of synthetic alternatives, including potential environmental harm, and the current labor-intensive nature of silk production using silkworm moths.

How did we decide on our project?

Our team began the ideation process for the 2023 iGEM season during March-April 2023, shortly after new members join. In our first round of ideation, we split into several groups and presented our ideas at our team general body meeting. We then narrowed these ideas down to four finalists via a survey of the entire team's preferences.

In the second phase of ideation, all team members chose one of the four finalists to join and prepared a presentation to our faculty sponsors, Drs. Dunleavy and Timp at the Department of Biomedical Engineering. The two faculty sponsors reviewed each idea and scored it on various factors such as feasability and safety.

On April 9th, 2023, we officially found out which idea we were going to pursue as our project for the 2023 competition season. The group that received the highest score presented the idea of biomanufacturing caddisfly silk in a bacterial chassis with the application of producing safe, biodegradable waterproof adhesives. Coming in at a close second was a project that would silence antibiotic resistance via targeted conjugation to gut microbiota, which was dropped by our faculty reviewers due to iGEM safety rules concerns. Rounding out the top four were projects focused on bioremediation of diesel pollutants and diagnostic kits for toxic shock syndrome.

What are the problems with current waterproof adhesives?

Adhesives in aquatic and wet environments have a wide variety of applications. In industry, they are utilized in ocean exploration and maintenance of water vehicles, while in the medical field they have potential applications ranging from closing incisions in orthopedic surgery [1] to filaments for sutures in oral surgery.

However, synthetic waterproof coating and underwater adhesives release PFAS (perfluoroalkanes) and other harmful chemicals into the environment, posing potential danger to humans and the environment. In particular, PFAS have gained attention for their toxicity and persistence, as they are capable of remaining in the environment for thousands of years before finally degrading. Thus, there is currently a demand for safe, biodegradable, and waterproof adhesives with no cytotoxic properties.

Caddisflies are winged insects as adults, but they spend much of their larval stage underwater in creeks and streams. The caddisfly larvae produce the silk protein fibroin, which is a heterodimer consisting of a heavy chain and a light chain. The protein is initially suspended in a liquid feedstock inside silk glands near the larva’s mouth. The liquid feedstock is then extruded through the opening of the gland, which causes the protein to undergo a phase transition that converts it into a solid silk fiber. Currently, the biochemical mechanisms underlying this transition are not fully understood. Finally, in the creek water, the silk gradually matures in a process known as “redenning”.

(Figure 1): (A) A casemaker caddisfly with a case made up of glass beads. (B-E) Increasing magnification of the silk inside each glass bead.

Caddisfly silk forms networks of fibers that adhere well to small objects such as twigs and pebbles, from which the caddisfly larvae construct protective structures by gluing them together. The silk is resistant to high temperatures up to 242 °C, resilient to deformation, and not cytotoxic to human cells. Most importantly, the silk is water-resistant and highly durable. These properties of caddisfly silk make it a promising candidate for use as an adhesive in wet environments.

What makes caddisfly silk unique?

While there has been relatively more research conducted on silk produced by spiders and silkworms, these fibers are not themselves adhesive, but are coated in a glycoprotein glue that is easily removed using warm water. This glue dissolves when exposed to water, making spider and silkworm silk ineffective for use as an underwater adhesive.

In contrast, caddisfly silk protein is naturally sticky and remains so in wet environments due to a large number of phosphorylated serine-rich repeats in the protein sequence. Once the silk protein is extruded from the gland, the negatively charged phosphorylated serine residues form complexes with positively charged calcium ions in the creek water [2].

Furthermore, as opposed to most underwater adhesives which must be applied before submersion, caddisfly silk adheres to materials that are already wet by displacing water from the surface of objects, and it can even adhere to biofouled objects (i.e. objects covered in algae and biofilms).

(Figure 2): A model of the silk spinning process within a caddisfly.

Why use synthetic biology to produce caddisfly silk?

The advantages and versatility of caddisfly silk make it an attractive candidate for developing a natural underwater adhesive, as opposed to the silk produced by other arthropods. However, it is impossible to farm caddisflies directly for their silk on an industrial scale as is done with silkworms, as the latter have been domesticated and selectively bred for silk production for millenia while caddisflies have not been altered by humans for silk production purposes. Furthermore, industrial silkworm silk production is highly labor-intensive, with workers often in poor conditions. Thus, the most practical and scalable method of producing caddisfly silk would be by expressing the silk protein in a bacterial chassis.

Using synthetic biology to produce caddisfly silk in bacteria enables the development of a simpler and more scalable method of production as opposed to trying to produce silk from live caddisflies. The caddisfly fibroin genes can be isolated and assembled into a plasmid with appropriate parts for gene expression, using a molecular cloning method such as golden braid. The plasmids can then be transformed into the final bacterial chassis, which can be grown in a bioreactor. The appropriate steps must be taken to ensure that the fibroin protein is phosphorylated, as in contrast to eukaryotes, bacteria seldom perform post-translational protein modifications.

Once the bacterial cells are expressing the genes on the plasmid, the fibroin protein can be extracted and purified by lysing and homogenizing the cells, then running the lysate through a chromatography column. A histidine tag can be used to isolate the fibroin protein via nickel affinity chromatography.

The protein product will ideally be of a form that can be stored for future use, and can hopefully be extruded into silk fibers using a device that mimics the mechanical stress required for inducing the phase transition from liquid to solid. We would also test the effects of exposure of the silk to calcium-rich water, and possibly perform measurements of silk fiber strength, adhesion capabilities, and water-resistant properties.

Although the process of producing natural caddisfly silk at an industrial would take years to refine, synthetic analogues of caddisfly silk would likely emit molecules that are damaging to the environment, and the production of such a material at an industrial scale may involve the use of environmentally damaging reagents and waste chemicals that can be difficult to store and dispose of safely. Developing a method to produce natural adhesives would be worthwhile when considering the possible drawbacks of synthetic polymer products and elimination of the need for human manual labor.

Inspiration from past iGEM teams

We were inspired by two past iGEM teams that successfully produced silk using a bacterial chassis. We hope to build on their work to develop a method of producing caddisfly silk with transgenic bacteria.

Here are additional iGEM teams that inspired our methods, creativity, and more.