HTGAA 2026: Individual Final Project Documentation

FINAL PRESENTATION SLIDES

SECTION 1: ABSTRACT

MycoBoard addresses the global e-waste crisis by engineering the fungi Neurospora crassa to grow biodegradable hyphal mats that function as breadboard-like electronic substrates. Conventional FR4 fiberglass PCBs contribute to the 62 million metric tons of annual e-waste, most of which is non-recyclable. MycoBoard leverages fungal thigmotropism to form conductive tracks within molded mats, with cmt metallothionein overexpression driving copper ion capture along the hyphae walls of N.crassa. The purpose is to create compostable electronic substrates that can break down in soil within weeks. The principle, validated by a Benchling-designed linear cassette and literature-based copper-loading estimates, states that engineered N. crassa could both biosorb copper and form conductive pathways via filamentous formation of hyphae. Aim 1 designs and validates a linear cassette construct for copper-responsive expression of cmt. Aim 2 transforms N. crassa experimentally, grows mats in breadboard molds, and tests LED circuit conductivity. Aim 3 replaces molds with optogenetic cmt control for pattern-directed copper deposition. Methods include Benchling DNA design, Twist synthesis, spheroplast electroporation, fungal mat cultivation, and multimeter/resistance validation.

SECTION 2: PROJECT AIMS

Aim 1 - Experimental Aim (executed):

The first aim of my final project is to design and computationally validate a Pccg-1=cmt-HA=hygR linear cassette for Neurospora crassa overexpression of cmt protein, using Benchling for sequence assembly, codon optimization, HA-tag fusion verification, and Twist order preparation. Methods include FungiDB for gene and promoter search, NEB Cutter for restriction site analysis, and literature-based copper biosorption estimations and predictions by N. crassa. The outcome is a complete Benchling file with verified reading frame and theoretical copper capture benchmark data of concentration of copper in a specific area of the fungal mat.

Aim 2 - Development Aim:

Translate the validated construct into a physical prototype by experimentally transforming N. crassa through spheroplast electroporation, then growing engineered mats in laser-cut/3D-printed breadboard-like molds, soaking in copper medium, and validating conductivity with a multimeter-powered LED circuit. This extends Aim 1 by testing hyphal thigmotropism first hand to track formation and copper deposition on the fungal mat for electrical conductivity of the material.

Aim 3 - Visionary Aim:

Replace physical molds with optogenetic control of cmt expression through a light-inducible promoter, allowing users to define custom circuit layouts that direct copper deposition during fungal growth. This realizes unique and on-demand compostable PCBs grown from spores, avoiding FR4-related pollution and enabling sustainable electronics production at scale.

SECTION 3: BACKGROUND

Beltramini & Lerch (1986) characterized Neurospora crassa copper metallothionein (cmt) as a low-molecular-weight protein binding 6 Cu(I) ions per molecule (Mr = 2200), induced by Cu(II) stress without any necessary oxidative action required. The cmt protein forms a Cu(I)-thiolate cluster homologous to vertebrate MTs, supporting metal homeostasis. Subramanyam et al. (1983) and further researchers showed copper toxicity produces blue mycelium with 8 mg Cu/100 mg dry weight and cell walls containing 12% copper, indicating successful and important copper biosorption capacity.

MycoBoard’s novelty is repurposing N. crassa’s natural copper-response pathway and biosorption capacity for conductive track formation in fungal mats, creating breadboard-like electronic substrates that could be compostable. This project integrates genetic engineering and thigmotropism to achieve custom-patterned metal deposition along the fungal mat. It expands synthetic biology by working with less common organisms such as N.crassa to robust fungal materials for electronics.

This project and similar existing proposals could help tackle the 62 million metric tons of annual e-waste, 80% of which is non-recyclable PCBs (Baldé et al., 2024). Biodegradable substrates could reduce landfill burden and explore more localized production. It mixes bioremediation, electronics and synthetic biology. Successful outcomes for MycoBoard would also further validate fungi as editable electronic chassis.

Even though MycoBoard involves genetic engineering for a beneficial and non-maleficient cause, copper or silver biosorption could mobilize metals if the fungal mats decompose improperly. Ethical considerations are key when working with living ecosystems and heavy metals. Possible containment methods for this could be auxotrophic markers, kill-switches, and lab-only strains that could prevent environmental release. Additionally to these, a key rule would be metal-loading protocols that use non-toxic concentrations. Throughout aim 2 and 3 future developments, iterative soil degradation assays are required to better understand copper toxicity thresholds and decomposition processes of the mats.

After presenting my research proposal during the Global Committed Listener presentation session, I received very useful feedback along with valuable references that helped me further understand MycoBoard’s positioning and technical framing (thank you to those who took the time to share these resources!). One particularly relevant publication by Rivnay et al. (2025) discusses how bioelectronics often requires high power and lacks the specificity and adaptability of some cells and tissues, and discussing that parallel advances in synthetic biology, biomaterials, and bioelectronics enable new opportunities in devices for regulated cell therapies, diagnostic tools, and next-generation robotics through biohybrid systems. Additionally, Lazaro-Vasquez and Vega (2019) demonstrated the use of mycelium composites with common digital fabrication techniques to replace plastic in electronics, specifically for inserting electronics in mycelium boards and making enclosures for electronics. However, their work focused only on enclosures and did not replace other components within the electronics; this is a gap that MycoBoard aims to address by engineering the fungal mat itself to form conductive tracks. On the other hand, some interesting advances are being done by The Rivnay Group’s work on organic bioelectronic materials that enable mixed ionic-electronic conduction for sensing and stimulation in biomedical settings (Rivnay Group, n.d.); and the Light Plate Apparatus (LPA), which could offer a potential platform for precisely controlling light-inducible expression of copper uptake and metallothionein genes in MycoBoard’s genetically engineered strains.

SECTION 4: EXPERIMENTAL DESIGN, TECHNIQUES, TOOLS, AND TECHNOLOGY

Detailed Plan for Aim 1 (Timeline: 2 weeks)

Techniques relevant to this project

• Lab Safety
• Bioethical Considerations
• DNA Construct Design
• Databases (e.g., GenBank, NCBI, Ensembl, and UCSC Genome Browser)
• Designing a Twist Order
• Use of Benchling
• Models and Notebooks
• Databases

1. Benchling DNA Construct Design: I used Benchling to assemble the linear cassette, by importing cmt CDS and Pccg-1 promoter from FungiDB, fusing C-terminal HA tag, and integrating hygR selection (see complete procedure here: https://pages.htgaa.org/2026a/sara-gaviria-escobar/projects/individual-final-project/index.html) . Annotation verified promoter-gene-terminator flow, codon optimization and translation viewer confirmed in-frame HA. The use of Benchling was key to validate the genetic logic for the cmt copper-responsive expression.

2. Twist Order Design:

The 4.5 kb Benchling cassette was longer than Twist’s single-fragment limit, so I split it into almost equal 2.4 kb and 2.1 kb pieces, keeping overhangs for future PCR or electroporation. This cost optimization avoided unnecessary backbone, targeting direct and easier spheroplast transformation for future development of the project.

How To Grow (Almost) Anything Industry Council companies associated with this final project

• Twist Biosciences/Ginkgo Bioworks (DNA synthesis)
• New England Biolabs (restriction enzyme analysis)
• Mycoworks (fungal materials precedent)

SECTION 5: Results & Quantitative Expectations

I chose to validate the DNA construct design for cmt overexpression in N. crassa by creating a complete Benchling cassette, checking reading frames and annotations, analyzing restriction sites, and deriving a literature-based copper-loading benchmark.

A detailed protocol for my validations:

1. Obtained cmt (NCU05561) and Pccg-1 from FungiDB.
2. Assembled Pccg-1=cmt-HA=hygR linear cassette.
3. Codon-optimized cmt, verified HA fusion translation.
4. Ran NEB Cutter to check there were no restriction enzyme sites. Results show no HA sites, 2 cmt sites (Eco0109I, MfeI), and multiple hygR sites.
5. Simulated a Twist order ($359.92).
6. Calculated Cu loading by researching experimental data in scientific literature.

The techniques for this protocol required:

• DNA Construct Design (Benchling assembly)
• Databases (FungiDB)
• Twist Order Design
• Models/Notebooks (Cu-loading calculation)
• Restriction Enzyme Analysis

The data obtained for validation was calculated based on existing scientific literature, and theoretical Cu biosorption values were calculated: Cu6 stoichiometry and 8% w/w value. Analysis also shows 54% dry mass as saturated cmt to match observed Cu, indicating broader biosorption contribution.

An unexpected challenge for this section was no direct cmt mg/cm² data found, which was resolved by using published stress-response values as upper bounds. The limitation, however, is that the estimate assumes uniform loading, which means future Western blots or similar methods are needed for cmt fraction validation.

To determine whether 0.8 mg of copper per cm² (measured from a fungal mat decomposing on soil according to scientific literature) falls below or above the toxicity threshold, the concentration must be converted to the standard unit of mg Cu per kg of dry soil. This calculation also assumes the fungal mat will decompose in the top 10 cm of soil (which is the typical active microbial zone) with a bulk density of approximately 1.3 g/cm³, 0.8 mg Cu/cm² over 10 cm depth equals a soil concentration of approximately 61.5 mg/kg (because 0.8 mg Cu ÷ 10 cm³ soil × 1.3 g/cm³ = 13 g soil = 0.013 kg; 0.8 / 0.013 ≈ 61.5 mg/kg). When compared to the cited thresholds, Shaw et al. (2020) observed loss of soil microbial functionality and community shifts above 200 mg Cu/kg, with no particular functional loss at or below 200 mg/kg. On the other hand, Rooney et al. (2006) reported plant toxicity EC50 values ranging from 36–536 mg/kg (in barley plants) and 22–851 mg/kg (in tomato plants) depending on soil properties, meaning effects typically emerge at concentrations higher than 60 mg/kg. Additionally, Caetano et al. (2016) derived soil screening values of 26.3–31.8 mg/kg for Cu based on multiple species and endpoints, no adverse effects are expected below these thresholds.

In conclusion, 61.5 mg/kg exceeds the preliminary screening value of ~30 mg/kg from Caetano et al. (2016) but is well below the 200 mg/kg functional threshold from Shaw et al. (2020) and the lowest plant EC50 values from Rooney et al. (2006). This means that while 0.8 mg/cm² is above the most conservative reference from my research, it remains under the threshold for significant microbial functionality loss (Shaw et al., 2020) and plant toxicity for most soils (Rooney et al., 2006). The fungal mat decomposition would not exceed the European Union’s agricultural warning limit of 200 mg/kg, but still needs further assessment depending on real copper concentrations from the actual mat grown at the lab.

SECTION 6: ADDITIONAL INFORMATION

References:

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Caetano, A. L., Marques, C. R., Gonçalves, F., Da Silva, E. F., & Pereira, R. (2016). Copper toxicity in a natural reference soil: ecotoxicological data for the derivation of preliminary soil screening values. Ecotoxicology, 25(1), 163-177.

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Supply list:

TOTAL: approx. 610 $