Project Description

What is Caddisfly Silk?

Caddisfly silk is secreted by the larval stage of animals within the order Trichoptera and is used to make nets, cases, and cocoons. This silk, while similar to silkworm silk, differs in one key way: underwater adhesion. Caddisfly silk is capable of adhering to already submerged objects, making it an ideal material for applications in wet environments. This silk protein is a heterodimer with a heavy chain and a light chain. The heavy chain is a large protein with many repeats.

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 interacts with naturally existing Ca2+ ions to begin the curing process and gradually matures in a process known as “redenning”.

How did we develop our project? (How is it different)

Building on the work of the 2023 Hopkins team, this year’s iGEM team sought to not only conduct further research on the base manufacturing process, but also focus on specific applications of caddisfly silk. We recognized that there was a lot of potential in advancing studies in this area, particularly because it covers a niche which isn’t entirely addressed by synthetic spider silk producers. Our project this year focuses on the development of a bio-inspired silk mesh with use cases as large as fishing nets to use cases as grafts and meshes within the human body. Throughout the year, we explored different ways to apply our product, speaking to stakeholders and implementing feedback.

Our aim is to continue working towards expression of caddisfly silk in yeast. Caddisfly silk is a heterodimer consisting of both a light and heavy chain fibroin (abbreviated L and H-fibroin, respectively), the former of which is relatively easy to express at ~800 base pairs. The real obstacle to caddisfly silk synthesis is expression of the H-fibroin protein, which is not only extremely long (19kb), but highly repetitive. Our group approached these caveats via two methods: 1. PCR the gene out using primers that would anneal to less repetitive sections in the middle of the gene, and 2. Synthesize the entire gene through a novel method that would stitch together motifs until the full gene was synthesized.

We believe synthetic biology is the key to developing such biomaterials because it allows for production to be easily scaled and leads to lower environmental impact as we shift towards renewable energy resources.

What are the issues with current bio-meshes?

Surgical meshes are used widely to support weakened tissue in cosmetic surgeries and hernia. These are either made from synthetic materials or animal tissue and can either be absorbable or non-absorbable. Non-absorbable mesh will still degrade over time and according to the FDA, most hernia surgeries involve the usage of meshes. The vast majority of meshes are made from polypropylene, which does not integrate into the existing cellular architecture well and can lead to implant induced illness. Its main advantages are that it is cheap, easily scaled, and has an easy procedure for insertion.

However, our work on CaddisGraft shows that we can satisfy these advantages while still being absorbable without further increasing the levels of microplastics in people. Our mesh design can be easily scaled and we have developed an extruder to easily apply and customize the mesh for each individual patient. We developed a business plan for our product.

Why Synthetic Biology?

Synthetic biology is the best way to approach this problem as purely biological materials require the usage of live animals and are incredibly expensive and synthetic materials are made from inorganic materials which can be harmful to humans. Synthetic biology allows for the benefits of these two approaches to be blended together in a way that can easily be scaled up. It is much easier, and more ethical, to synthesize caddisfly silk from yeast cells than from the bugs themselves. Additionally, it is very labor intensive and low yield to try and extract caddisfly silk from larvae and simply not a sustainable practice.

Inspiration

We took inspiration for the multi-silk design from the U-Copenhagen team’s project Netlantis for the scaffolding of our graft design and built off of the work of the 2023 Hopkins iGEM team on Caddissilk. We are however only attempting to express caddisfly silk- not silkworm silk as well. We are using a different synthesis method this year in order to be able to achieve a higher number of fibroin repeat sequences.

Additionally, some other iGEM teams whose work we found interesting and useful to this project include:

Proposed Workflow

Overall Workflow for Caddisgraft. Creation of a H-fibroin and L-fibroin yeast plasmids → transformation of plasmids into yeast → protein purification (HPLC) → extrusion of purified silk protein with glue gun → creation of silk mesh → application to cosmetic surgery

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Dr. Haiquan Mao at Johns Hopkins University said, “you want the material to be porous enough to allow host tissue to grow into it but also retain its shape and the integrity of the repair site, and not lead to scarring and fibrosis by host immune cells.”