Ermine Moth Caterpillar Silk Matrices as Biofilters for Airborne PFAS Particles

In the summer of 2025, I first encountered a tree infested with the intricate webbings spun by Ermine moth caterpillars in Amsterdam’s Vondelpark. It’s an eerie sight: the tree stripped entirely of foliage and shrouded in silver-white silk, with hundreds of caterpillars crawling across the delicate architecture. As a biodesigner, though, it’s a thrilling display of collective behavior crafting biological structures. While the tree stands leafless for a time, this temporary infestation leaves no lasting harm to healthy specimens. The silk naturally disintegrates within months, recycling nutrients back to the soil.

These silk networks boast remarkable structural qualities: they are comprised of ultrafine yet resilient threads that endure wind and rain  and their intricate matrix yields a vast aerially exposed surface area, maximizing atmospheric contact.  (Volenikova et al., 2022)

At first, the silk’s exceptional tensile strength inspired visions of potential application of the silk as a biomaterial. However, this would be a reductionist approach to this complex mechanism, as it negates qualities like architectural capacity and ecological context. Could we instead augment this living system with minimal disruption, rather than extracting it for human designs?

Nature is inherently non-sterile, as are Ermine moth caterpillars, whose bodies teem with symbiotic microbes. As the caterpillars navigate their web, these bacteria disperse across the matrix—positioning colonies in direct aerial contact. This setup ideally suits metabolic processes optimized for airborne exposure, such as enzymatic pollutant breakdown.

This brings me to my overarching research question: Can symbiotic bacteria from Ermine moth caterpillars, engineered with PFAS-degrading enzymes, bioremediate airborne PFAS when deployed on their native silk matrices?

PFAS, per- and polyfluoroalkyl substances, commonly known as “forever chemicals” are a group of chemical compounds that have a significant negative effect on human and ecological health. (Stanford Medicine, 2024) On a personal note, I grew up in ‘de Alblasserwaard’, a region within the province of Zuid-Holland, the Netherlands. In recent years, it’s become known that the entire area is heavily contaminated with PFAS originating from  industrial emissions by a local DuPont factory. (Dordt Centraal, 2023) This will likely imply that my health and the health of my family will be negatively impacted as a result of living in a polluted environment. 

On a hopeful note, a paper published in early 2025 entitled “PFAS biodegradation by Labrys portucalensis F11: Evidence of chain shortening and identification of metabolites of PFOS” describes promising evidence that a bacterial species isolated from PFAS contaminated soil in Portugal managed to enzymatically biodegrade  PFOS, 6:2 FTS, and 5:3 FTCA into shorter-chain compounds. (Wijayahena et al., 2025) 

Initially I formulated my three aims for the project as follows: 

• Aim 1 - Synthesizing  a biosensor for BmCoc enzymatic degradation of Sericin-2 utilising GFP signaling and testing it on Ermine moth caterpillar silk sourced from field research. 

A key component of the envisioned genetic circuit in aim 2 is the capacity to degrade the silk protein  sericin-2 as a carbon source in conditions when PFAS particles are not readily available.

• Aim 2 - Engineering an initial bacterial chassis with a genetic circuit consisting of three genetic components:

  • A PFAS detecting biosensor (Mann, et al. 2023)

  • A gene that encodes for an enzyme that breaks down Sericin 2 (Rodbumrer, et al. 2012) and 

  • A gene that encodes for enzymatic degradation of PFOS, 6:2 FTS, and 5:3 FTCA into shorter-chain compounds (Wijayahena et al., 2025) 

Due to their toxic nature, PFAS particles would be expelled by the bacterial chasis. By deleting the gene that encodes for the Tol-c efflux membrane protein, intracellular accumulation of particles would then facilitate the detection and degradation of these compounds. 

• Aim 3 - Introducing the strategy into local affected ecosystems in the Netherlands, as a non-disruptive mechanism for bioremediation, after rigorous testing to meet EU ecological risk assessment regulations (ERAs) and conducting EU mandated post-market environmental monitoring (PMEM). 

However, after reconsideration, I came to the conclusion that for my aim 1, a biosensor that utilised GFP signaling would only tell me whether the gene of interest was successfully expressed, and it would give a quantifiable indication of the protein of interest. This would not give me any data on the actual degradation of the silk or the proliferation of bacterial colonies. The plasmid design would be relatively expensive for the potential data it would yield.

Therefore I decided to simplify my plasmid and focus on developing a simple, but effective experimental protocol that would give me data on all three of these variables. 

Complete Detailed Experimental Protocol: Ermine Moth Silk Degradation by Engineered E. coli*

Objective   Demonstrate that E. coli expressing BmCoc (Bombyx mori cocoonase) can degrade sericin from wild ermine moth silk (weight loss), proliferate using sericin as a nutrient source (CFU count), and secrete functional His-tagged enzyme (protein quantification).

Experimental Design

15 wild silk fibers (n=3 per assay/condition):

Timeline:

Day 0: Preparation
1. Harvest and Sterilize Wild Silk 
"Collect mature ermine moth (Yponomeuta padellus) silk webs or cocoons from infested trees. Carefully dissect ~1 cm silk fibers, removing debris. Place 15 fibers in a sterile 15 mL tube with 10 mL 70% ethanol and rock gently for 15 minutes at room temperature. Decant ethanol and rinse fibers three times with 10 mL sterile PBS (pH 8.0), 5 minutes each rinse. Transfer to a new sterile Eppendorf tube and air dry completely in a biosafety cabinet for 30 minutes."

2. Initial Weight Measurements

"Working in a biosafety cabinet, tare an analytical balance with pre-weighed filter paper and Eppendorf tube. Place one dry silk fiber on the filter paper and record the DRY_START weight (target 9-11 mg per fiber) for fibers 1-15. Store weighed fibers in a sterile tube."

3. Start E. coli Overnight Cultures (15 min)

"Pick a single colony of BmCoc+ E. coli (pET28-BmCoc-His, AmpR) and a single colony of empty vector control into separate 5 mL LB + 50 µg/mL ampicillin tubes. Incubate overnight at 37°C, 200 rpm shaking."

Day 1: Culture Preparation & Silk Inoculation
4. Grow Day Cultures 

"The next morning, dilute each overnight culture 1:100 into 50 mL fresh LB + Amp in 250 mL flasks. Grow at 37°C, 200 rpm until OD600 = 0.5 (approximately 3 hours). For BmCoc+ culture only, add 0.5 mM IPTG and induce for 4 additional hours at 30°C (reduces inclusion bodies)."

5. Prepare Silk Incubation Medium
"Prepare sterile minimal medium: PBS pH 8.0 + 0.2% glucose + 0.1% casamino acids + 50 µg/mL ampicillin. This forces cells to use sericin as primary nutrient source during 48h incubation."

6. Inoculate Silk Fibers 

"In a 24-well plate, add 2 mL OD600=0.5 culture per well containing one sterile silk fiber (fibers 1-12). For no-cell controls (fibers 13-15), add 2 mL minimal medium only. Save 1 mL culture from each condition in Eppendorf tubes for supernatant analysis. Incubate at 37°C, 200 rpm shaking for 48 hours."

Day 3: Sample Harvesting 

7. Weight Loss Measurement (Fibers 1-15)

"Using sterile tweezers, carefully remove each silk fiber from its well. Transfer to a clean Petri dish and wash by dipping three times in 10 mL PBS (5 seconds each dip with gentle swirling). Perform one brief 3-second MilliQ water rinse to remove salts. Blot excess water on Kimwipe for 10 seconds. Transfer to pre-weighed filter paper + Eppendorf tube. Air dry 30 minutes at room temperature in a desiccator, then oven dry at 60°C for 1 hour. Cool 30 minutes in desiccator and record DRY_END weight."

8. CFU Proliferation Assay (Fibers 4, 6, 10, 12)

"Place one CFU fiber in a 1.5 mL Eppendorf with 900 µL PBS + 0.1% Tween20. Vortex vigorously for 2 minutes to detach biofilm cells. Transfer 100 µL to new tube with 900 µL PBS (10⁻¹ dilution). Repeat serial dilutions to 10⁻⁵. Plate 100 µL of 10⁻⁴ and 10⁻⁵ dilutions on LB + Amp agar plates using sterile spreaders. Incubate plates overnight at 37°C."

9. Supernatant Collection for Protein Analysis 
"Centrifuge 1 mL saved cultures from each condition at 16,000g for 5 minutes. Transfer supernatant to new Eppendorf tubes and store at 4°C for His-tag purification."

Day 4: Analysis 

10. Colony Counting

"Count colonies on plates with 30-300 colonies (ideal range). Calculate CFU per fiber = (colonies counted) × dilution factor × 10."

11. His-Tag Protein Purification

"To 1 mL supernatant, add 20 µL Ni-NTA slurry. Nutate 1 hour at room temperature. Spin 1 minute, discard supernatant. Wash beads three times with 500 µL wash buffer (50 mM phosphate pH 8.0, 300 mM NaCl, 20 mM imidazole). Elute twice with 50 µL elution buffer (same + 300 mM imidazole). Pool eluates for quantification."

12. Protein Quantification 

"Bradford assay: Mix 2 µL eluate + 200 µL Bradford reagent. Read OD595 after 10 minutes vs BSA standards. A280 confirmation: Measure 1 µL eluate directly on NanoDrop using BmCoc extinction coefficient."

Day 5: Data Analysis & Imaging

13. Calculate Results

% Weight loss = 100 × (DRY_START - DRY_END) / DRY_START

Expected: BmCoc+ = 15-25%, Uninduced = 1-3%, No cells = <1%

14. Optional Imaging

"Image representative fibers pre/post treatment using stereo microscope. Stain one spare fiber with 0.1% Crystal Violet for total biomass visualization."

Transform into e.coli chassis. Experiments involve exposure to wild Ermine caterpillar moth silk. Silk samples are inoculated with both transformed and uninduced control, accompanied by non-cell baseline. Samples are then analysed in 3 ways: 

1. Weight loss measurement of silk

2. CFU proliferation assay 

3. Protein quantification through Bradford assay