The Profound Uses of DNA Engineering between Spiders and Silkworms

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Spider silk fibres produced by silkworms Junpeng Mi, Donghua University

Silk, an ancient fabric, has transcended civilisations and so, in the constant quest for stronger, more resilient materials, scientists have turned to the wonders of science, particularly genetic engineering. One of the most remarkable discoveries in this front of science is the fusion of spider DNA with silkworms to produce silk of unparalleled strength and durability. This groundbreaking achievement not only promises to revolutionise textile production but also highlights the power of genetic manipulation in shaping materials in the future.

Traditionally, silk was harvested from silkworms (Bombyx mori), a process that relied on the natural secretion of silk proteins by the larvae. Let’s look into that

Silk Production

Silk production in silkworms is a fascinating biological process that involves a complex interplay of genetic, biochemical, and physiological mechanisms. Here’s a detailed look at the deep biology behind silk formation:

1. Genetics and Proteins Involved:

Silk is primarily composed of two proteins: fibroin and sericin. Fibroin is the core protein of silk, making up around 75% of its structure. It is composed of heavy and light chains. The heavy chain, H-fibroin is about 350 kilodaltons, while the light chain, L-fibroin is around 25 kilodaltons. These chains are linked by disulfide bonds. The fibroin gene family is highly conserved and specifically expressed in the silk gland cells of silkworms. Sericin is a protein that acts as a glue to hold the fibroin fibres together. It makes up about 25% of the silk and is interestingly soluble in hot water. Sericin is encoded by multiple genes that are rich in serine and other amino acids.

2. Silk Gland Structure and Function:

The silk glands of silkworms are specialised organs divided into three main parts: Anterior Silk Gland: Responsible for the secretion of sericin. Middle Silk Gland: Produces fibroin. Posterior Silk Gland: Stores and synthesises the silk proteins.

3. Silk Protein Synthesis

Silk proteins are synthesised in the posterior silk gland cells, which are highly active in protein synthesis. These cells contain high amounts of Rough Endoplasmic Reticulum and Golgi apparatus within the cells, reflecting their role in producing and processing large amounts of protein.

4. Protein Processing and Storage:

Once synthesised, fibroin and sericin are transported to the middle and anterior silk glands. Here, they are stored in a highly concentrated and hydrated form. The proteins are kept in a liquid crystalline state, which is essential for their eventual conversion into solid silk fibres.

5. Secretion and Fiber Formation:

When the silkworm is ready to spin its cocoon, the stored proteins are transported to the spinneret. During this process the transition from liquid to solid occurs. As the silk proteins pass through the narrow ducts of the silk glands, changes in pH, calcium ion concentration, and very strong forces trigger the transition from a liquid crystalline state to solid fibres. Next, the fibroin molecules align themselves in an orderly fashion, leading to the formation of β-sheet structures, which are characteristic of silk’s crystalline regions. These regions provide tensile strength and durability. As the fibroin fibres emerge from the spinneret, they are coated with sericin, which solidifies upon exposure to air, helping to glue the fibres together and therefore provides a protective coating.

6. Biochemical Environment:

Several factors influence the silk spinning process: For example, the pH gradient in the silk gland changes from acidic to neutral, which helps in the assembly and solidification of the silk. High concentrations of potassium, phosphate, and calcium ions play very important roles in maintaining the stability and proper assembly of silk proteins. The hydration state of the silk proteins is highly regulated, ensuring they remain soluble until the final stages of fibre formation.

7. Molecular Mechanisms:

At the molecular level, the self-assembly of fibroin into fibres involves the formation of micellar structures. These micelles align and elongate, forming larger fibrils, which then bundle together to create the final silk fibre. This process is guided by hydrophobic interactions, hydrogen bonding, and β-sheet formation.

8. Regulatory Mechanisms:

Silk production is under tight genetic and hormonal control. Ecdysteroids and juvenile hormones regulate the timing of silk protein synthesis and secretion, ensuring that silk production is synchronised with the larval stages of the silkworm’s development.

Silk production in silkworms is a remarkable example of biological engineering. It involves sophisticated genetic programming, specialised glandular structures, and precise biochemical conditions to produce one of nature’s most extraordinary materials.

While conventional silk possesses mediocre strength, it pales in comparison to the remarkable resilience of spider silk.

Spider silk stands out as one of nature's most extraordinary materials. Renowned for its exceptional toughness and elasticity, spider silk has captivated scientists for decades. However, the challenges of harvesting silk from spiders are incredibly hard to cultivate and farm if you put them together they end up attacking each other due to their territorial and cannibalistic features, due to that reason large-scale industrial applications just aren't possible. This led researchers to explore alternative avenues, ultimately leading to the groundbreaking idea of splicing spider DNA with silkworms.

Before we delve into this, let's have a look at the production of spider silk.

Spider silk formation is a complex and highly specialised biological process that involves multiple silk glands, a variety of silk proteins, and intricate biochemical and mechanical mechanisms.

Genetics and Proteins Involved:

Spider silk is composed of a variety of proteins known as spidroins. Each type of silk is produced by different spidroins with distinct properties.

Major Ampullate Spidroin: is used for the dragline and frame threads. They are the primary proteins in dragline silk, providing high tensile strength and elasticity.

Minor Ampullate Spidroin: is used for temporary scaffolding during web construction. Flagelliform Spidroin: This protein is found in the capture spiral and is highly elastic. Tubuliform Spidroin: is used for egg sacs, providing structural support. Aciniform Spidroin: is used for wrapping prey and in web construction. Aggregate Spidroin: is the sticky silk used in the capture spiral for catching prey.

Silk Gland Structure and Function: Spiders have multiple silk glands, each specialised for producing a different type of silk. The main types of glands include: Major Ampullate Glands: Produce the dragline silk. Minor Ampullate Glands: Produce temporary scaffolding silk. Flagelliform Glands: Produce the elastic capture spiral silk. Tubuliform Glands: Produce egg sac silk. Aciniform Glands: Produce silk for wrapping prey and web construction. Aggregate Glands: Produce glue-like silk for sticky capture threads.

Silk Protein Synthesis Spidroins: They are synthesised in the silk gland cells, which are highly specialised for protein production. These cells contain abundant rough endoplasmic reticulum and Golgi apparatus within cells for protein synthesis.

Protein Processing and Storage: The spidroins are synthesised as large, repetitive proteins with specific domains that enable their assembly into fibres. They are stored in a highly concentrated, liquid crystalline state in the silk glands.

Secretion and Fibre Formation: When a spider spins silk, the stored liquid silk proteins are transported through the spinning duct to the spinnerets. During this process the transition from liquid to solid occurs. As the silk proteins pass through the narrow ducts, changes in pH, ion concentration, and many forces induce the transition from a liquid crystalline state to solid fibres. The spidroin molecules align into β-sheet crystals, which are interspersed with amorphous regions. The β-sheet crystals provide strength, while the amorphous regions confer elasticity. The spinnerets contain microscopic spigots that control the extrusion of silk. The spider can control the thickness and type of silk by adjusting the flow through different spigots.

Biochemical Environment: The spinning duct environment is crucial for silk formation. The pH gradient shifts from neutral to acidic, facilitating protein assembly. Calcium and phosphate ions play key roles in maintaining the solubility and promoting the solidification of spidroins. The water content is precisely regulated to ensure the silk proteins remain in a soluble state until extrusion.

Molecular Mechanisms: The assembly of spidroins into silk fibres involves: Nucleation and micelle formation where spidroins self-assemble into micellar structures within the gland. These micelles align and merge to form larger fibrils. Fibrils bundle together, transitioning from a liquid to a solid state as they pass through the spinning duct.

Regulatory Mechanisms: Silk production is tightly regulated by genetic and environmental factors. Hormonal signals and mechanical stress can influence the expression of spidroin genes and the activity of silk glands. The journey towards creating spider-silk enhanced silkworms began with a deep dive into the genetic makeup of both spiders and silkworms. Scientists meticulously mapped out the genes responsible for silk production in spiders, in this case, the East Asian orb-weaving spider (Arsneus Ventricosus), isolating the key sequences that encode the proteins responsible for the silk's strength and elasticity. These genes, when transplanted into the genetic code of silkworms, held the promise of giving the larvae the ability to synthesise spider silk proteins alongside their native silk.

Using advanced genetic engineering techniques, such as CRISPR, researchers like Junpeng Mi then introduced these spider silk genes into the genome of silkworms.

What is CRISPR? CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and is a revolutionary gene-editing technology that enables precise, directed changes to genomic DNA. It is based on a natural defence mechanism used by bacteria to protect against viral infections.

Background and Discovery:

CRISPR sequences were first discovered in the genomes of bacteria. These sequences are composed of short, repetitive DNA sequences with "spacers" in between, which are derived from previous exposures to viral DNA. The system allows bacteria to almost "remember" and defend against specific viruses.

Components of the CRISPR System:

The CRISPR system consists of two main components: Cas proteins: These are enzymes that can cut DNA. The most commonly used Cas protein in gene editing is Cas9.

Guide RNA: This gRNA molecule guides the Cas protein to the specific location in the genome where the cut should be made. The gRNA has two parts: A sequence that is complementary to the target DNA sequence and a scaffold sequence that binds to the Cas protein.

Mechanism of Action:

The CRISPR-Cas9 system works as follows:

Binding: The gRNA binds to the Cas9 protein, forming a complex.

Targeting: The gRNA-Cas9 complex searches the genome for a DNA sequence that matches the gRNA.

Cutting: Once the target DNA is found, the Cas9 protein makes a double-strand break in the DNA at that location.

Repair: The cell attempts to repair the break. There are two main types of repair: Non-homologous end joining (NHEJ): This method can introduce small insertions or deletions (indels) at the cut site, often leading to gene disruption.

Homology-directed repair: This method uses a template to repair the break, allowing for precise edits or the insertion of new sequences.

Applications:

CRISPR technology has a wide range of applications:

They can:

Disable specific genes to study their function Correct genetic mutations that cause disease

Develop crops with desirable traits such as disease resistance or improved yield

Potentially treat genetic disorders, cancer, and other diseases.

Create organisms with capabilities for biotech applications

Advantages:

CRISPR offers several advantages over previous gene-editing technologies:

Precision: It allows for highly specific targeting of DNA sequences.

Efficiency: It can introduce changes rapidly and at a relatively low cost.

Versatility: It can be used in a wide variety of organisms and cell types.

Challenges and Ethical Considerations:

While CRISPR has great potential, it also presents several challenges and ethical concerns. Unintended alterations to the genome can occur, which may have harmful consequences. In some cases, not all cells in an organism are edited uniformly. Issues such as gene editing in human embryos and the potential for "designer babies" raise significant ethical questions. There is ongoing debate about how CRISPR technology should be regulated to ensure safe and ethical use.

CRISPR is a very powerful and versatile gene-editing tool that has transformed biology and genetics. Its ability to make precise changes to DNA holds a lot for future advancements in medicine, agriculture, and biotechnology. However, consideration of its potential risks and ethical implications is essential as the technology continues to develop and be applied in various fields.

Transgenesis involves precisely inserting the desired genetic material into the silkworm embryos, ensuring that it would be passed down to future generations. One of the most remarkable aspects of this procedure is the seamless integration of spider DNA into the silkworm genome. Through careful manipulation at the molecular level, scientists were able to ensure that the spider silk genes would be expressed within the silk glands of the silkworms.

As the transgenic silkworms matured, they began to produce silk that was unlike anything seen before. The hybrid  silk, infused with the properties of spider silk, exhibited exceptional strength and durability. Tensile tests  revealed that it could withstand far greater stresses and strains; it's estimated to be ten times as strong as nylon , making it ideal for a wide range of applications. The implications of this breakthrough are incredible . In textiles, spider-silk enhanced silk opens up new possibilities for creating fabrics that are not only incredibly strong but also lightweight and flexible. From high-performance sportswear to industrial materials, the potential applications are virtually limitless.

Moreover, the biocompatibility of silk makes it well-suited for use in the medical field. The enhanced strength and durability of the spider-silk enhanced silk make it an ideal candidate for surgical sutures, wound dressings, and tissue engineering scaffolds. Additionally, its ability to degrade naturally within the body reduces the risk of adverse reactions, making it safer for use in medical devices and implants.

This new technology also reduces the toll conventional clothing manufacturing has on the environment significantly also due to its ability to degrade naturally, reducing the reliance on resource-intensive materials like cotton and synthetic fibres, thereby curbing pollution and conserving natural resources.

However, as with any technological advancement, the creation of spider-silk enhanced silkworms raises important ethical and environmental questions. Concerns have been raised about the potential impact of releasing transgenic organisms into the environment, as well as the implications of  manipulating the genetic makeup of living organisms. Furthermore, there are questions about the welfare of the transgenic silkworms themselves, and whether they may suffer any adverse effects as a result of the genetic modifications. It will be important for researchers to address these concerns and ensure that appropriate safeguards are in place to minimise any potential risks.

Looking to the Future

Despite these challenges, the creation of spider-silk enhanced silkworms represents a significant step forward in the field of materials science. By harnessing the power of genetic engineering, scientists have unlocked new possibilities for creating stronger, more resilient materials that could have far-reaching impacts across a wide range of industries. As researchers continue to refine their techniques and explore new applications, the future looks brighter than ever for spider-silk enhanced silk. From textiles to medicine and beyond, the possibilities are limited only by our imagination. In the coming years, we can expect to see even more remarkable advancements as we continue to harness the power of nature to create the materials of tomorrow.