Week 7 HW: hw-genetic-circuits-part-ii
Week 7 — Genetic Circuits Part II: Neuromorphic Circuits
Assignment Part 1: Intracellular Artificial Neural Networks (IANNs)
The shift from Boolean genetic circuits to Intercellular Artificial Neural Networks (IANNs) represents a move from simple digital logic to complex, analog, and adaptive biological computing.
Compared to traditional Boolean genetic circuits—such as standard AND, OR, or NOT gates—Integrated Analog Neural Networks (IANNs) offer distinct advantages for processing complex biological inputs. The primary limitation of Boolean logic lies in its “all-or-nothing” binary thresholding, which often results in significant information loss when dealing with environmental concentration gradients like toxin levels or nutrient density. In contrast, IANNs inherently support analog signal processing, enabling a continuous, graded response. By fine-tuning promoter strength or RBS efficiency, IANNs can assign specific weights to various environmental inputs ($X_1, X_2…$), empowering cells to prioritize certain signals over others during decision-making.
This architecture significantly alleviates the metabolic burden on the host; while a multi-input Boolean gate requires a large library of orthogonal transcription factors, the IANN framework allows multiple inputs to efficiently “fan-in” to a single regulatory node. Furthermore, by leveraging the Hill function—which serves as a biological equivalent to the sigmoidal activation functions in neural networks—IANNs effectively filter out molecular noise. This prevents the “flickering” issues common near the thresholds of Boolean circuits, substantially enhancing the robustness of genetic circuits within the complex intracellular environment.
“Smart” Precision Oncology
A compelling application for an IANN is a Selective Cancer-Cell Classifier.
Goal: An intracellular IANN that triggers “cell suicide” (apoptosis) only if a specific combination of microRNA (miRNA) biomarkers—unique to a specific cancer subtype—is detected.
Input/Output Behavior:Inputs ($X_n$): The inputs are the intracellular concentrations of 4–6 different miRNA biomarkers ($miRNA_{1…6}$). Some are “high” in cancer (positive weights), and some are “low” in cancer but “high” in healthy cells (negative weights/inhibitors).Processing: The IANN functions as a perceptron. It calculates the weighted sum of these miRNAs.Positive Weights: miRNAs that trigger the expression of a pro-apoptotic protein (e.g., BAX).Negative Weights: miRNAs that trigger an interlacing “decoy” or inhibitor (e.g., an antisense RNA or Csy4 to degrade the BAX mRNA).Output ($Y$): If the weighted sum exceeds a specific threshold (the “activation potential”), the cell produces enough pro-apoptotic protein to trigger programmed cell death. If the cell is healthy, the sum remains below the threshold, and the cell lives.
Limitations and Challenges:Metabolic Burden: Expressing the components of the IANN (the synthetic receptors, the processing RNAs, and the output proteins) consumes significant cellular energy (ATP and ribosomes), which might slow down the cell or lead to the circuit being “evolved out” (mutated) over time.Threshold “Leakiness”: Biological systems are rarely 100% “off.” Even in healthy cells, the IANN might produce trace amounts of the output protein. If the output is a potent toxin or cell-death trigger, even a tiny amount of “leakage” could kill healthy cells.Weight Precision: It is difficult to precisely “tune” biological weights. In a computer, a weight can be exactly $0.75$; in a cell, a “weight” depends on binding affinities ($K_d$) and protein decay rates, which fluctuate with temperature and cellular health.
Analysis of the Csy4-Regulated Perceptron
Analysis of the Csy4-Regulated PerceptronIn the diagram you described (the Csy4/Fluorescent Protein perceptron): X1 (Csy4 DNA): Acts as the Inhibitory Input. When $X1$ is transcribed and translated, the Csy4 endoribonuclease is produced. X2 (Reporter DNA): Acts as the Excitatory Input. It produces the mRNA for the fluorescent protein. Regulation (The “Logic”): The mRNA from $X2$ contains a specific RNA recognition site for Csy4.If Csy4 is present ($X1$ is HIGH), it cleaves the $X2$ mRNA, preventing translation of the fluorescent protein.If Csy4 is absent ($X1$ is LOW), the $X2$ mRNA remains intact and is translated into a fluorescent signal.
This setup functions as a Single-Layer Perceptron where the weight for $X1$ is negative. The final fluorescent output represents the state of the activation function after integrating the transcription/translation rates of both inputs.
+------------------ 第1层（隐藏层） ------------------+ +------------------ 第2层（输出层） ------------------+ | | | | | X1 DNA (编码Csy4) X2 DNA (编码转录因子) | | Y DNA (编码荧光蛋白) | | | | | | | | | +---- 转录(Tx) ----+ | | | +---- 转录(Tx) ----> mRNA(荧光蛋白) | | | | | | | | | v v | | | | | mRNA1 mRNA2 | | v | | | | | | 受内切酶E调控（切割） | | v v | | | | | 翻译(Tl) 翻译(Tl) | | v | | | | | | 剩余完整mRNA | | v v | | | | | TF1蛋白 TF2蛋白 | | v | | \ / | | 翻译(Tl) | | \ / | | | | | \/ | | v | | 结合启动子(内切酶基因) | | 荧光蛋白（输出） | | | | | | | v | | | | 转录(Tx) | | | | | | | | | v | | | | mRNA(内切酶) | | | | | | | | | v | | | | 翻译(Tl) | | | | | | | | | v | | | | 内切酶E（第1层输出）----------------------+--------------------------------------------------->| | | | | +------------------------------------------------------+ +------------------------------------------------------+
Brief working principle:
Layer 1: The two input DNAs (X1 and X2) are transcribed and translated to produce TF1 and TF2 proteins, respectively. These two proteins bind to the promoter and drive the expression of the endonuclease gene, ultimately outputting endonuclease E.
Layer 2: The input Y DNA is transcribed into fluorescent protein mRNA. This mRNA is cleaved (regulated) by endonuclease E output from Layer 1. The remaining intact mRNA is translated to produce the fluorescent protein output.
Assignment Part 2: Fungal Materials
Existing fungal materials: examples, applications, advantages and disadvantages
Mycelium-based composites (construction, packaging, furniture)
Mycelium-based composites (MBCs) are produced by growing fungal mycelium on agricultural substrates such as straw, wood chips, sawdust, or other lignocellulosic waste streams. After colonisation, the material can be heat-treated to stop fungal growth and processed into solid forms. These composites have emerged as sustainable alternatives to synthetic foams (e.g., polystyrene), engineered wood products, and even some plastics.
Applications: Building blocks, insulation panels, facade panels, door cores, flooring, cabinetry, protective packaging (as a replacement for expanded polystyrene), furniture, and sculptures.
| Advantage | Disadvantage |
|---|---|
| Low energy input for production — fungi grow on low-cost agricultural residues at ambient temperatures | Lower mechanical strength and load-bearing capacity compared to conventional timber or concrete — currently limited to temporary structures or non-structural applications |
| Fully biodegradable at end-of-life; no persistent microplastic pollution | Scalability challenges — producing large amounts of uniform material for industrial standards remains difficult |
| Superior fire resistance — mycelium composites exhibit low heat release, minimal smoke production, high char yield, and self-extinguishing properties compared to synthetic polymers like polystyrene | Sensitivity to moisture and water — requires treatment or coating for outdoor or high-humidity applications |
| Excellent acoustic absorption and low thermal conductivity (superior insulation performance compared to synthetic foams) | Currently higher unit cost at small production scales; cost competitiveness requires further scale-up |
| Carbon sequestration during growth — fungi absorb CO₂ as they grow, unlike plastic manufacturing which releases CO₂ | Inconsistent material properties due to biological variability among strains and cultivation conditions |
| Uses agricultural waste as feedstock, promoting circular economy principles | Regulatory hurdles — construction materials must meet strict building codes, making certification lengthy |
Mushroom-derived leather (mycelium leather)
Mycelium can be processed into flexible, leather-like sheets that serve as alternatives to both animal leather and synthetic faux leather (e.g., polyurethane). Researchers have developed techniques to produce such materials using split gill mushroom (Schizophyllum commune), Talaromyces sp., Pleurotus albidus, and Lentinus velutinus. Post-processing treatments (e.g., glycerol for flexibility, polyethylene glycol for stiffness) can tune the final properties.
Applications: Handbags, wallets, footwear, watch straps, car seat upholstery, steering wheel covers, and fashion accessories.
| Advantage | Disadvantage |
|---|---|
| Animal-free and ethically produced — no livestock suffering | Tensile and tear strength can be ~50% lower than genuine leather, requiring coatings or reinforcement for high-wear applications |
| Compostable at end-of-life — avoids landfill accumulation | Requires post-processing (coatings, crosslinkers) to achieve desired properties, adding cost and complexity |
| Avoids toxic chromium tanning used in conventional leather production, preventing river contamination | Production processes not yet fully standardised; material consistency varies with strain and substrate |
| Breathable and lighter than traditional leather | May still require synthetic polymer coatings (e.g., PVC, PLA) to enhance water resistance and durability |
| Tunable properties — genetic variations among strains can be leveraged to alter flexibility, water resistance, thickness, and suppleness without synthetic engineering | Scaling production to fashion industry volumes remains a challenge |
Biotextiles and flexible mycomaterials
Flexible mycomaterials are thin, textile-like sheets produced directly from fungal mycelium. These materials can be grown on lignocellulosic waste without needing animal inputs or petroleum.
Applications: Clothing, upholstery, technical textiles, and fashion items.
| Advantage | Disadvantage |
|---|---|
| Reduces reliance on water-intensive cotton farming and petroleum-based synthetic fibres | Mechanical strength may require polymeric coatings (e.g., PVA) for certain applications |
| Complete biodegradability compared to polyester and nylon | Thermal stability below that of many synthetic textiles without coating |
| Low environmental footprint across production cycle | Industrial-scale manufacturing capacity not yet established |
Fungal biomass for food proteins (precision fermentation)
Yeast and filamentous fungi are used as cell factories for producing high-value proteins through precision fermentation. These systems can secrete correctly folded, functional proteins directly into the culture medium, avoiding costly cell lysis and purification steps.
Applications: Animal-free dairy proteins (casein, whey), egg proteins, collagen, and other food proteins for alternative protein products.
| Advantage | Disadvantage |
|---|---|
| Secretion capability — fungi export proteins into the culture medium, lowering downstream processing costs compared to bacterial intracellular production | High-volume, low-margin products require gram-per-litre titres to be cost-competitive with conventional agriculture |
| Eukaryotic post-translational modifications (glycosylation, disulfide bonds, etc.) essential for functional food proteins | Precision-fermented food proteins still advancing from pilot to routine industrial manufacture |
| Can be grown on low-cost media, keeping production costs relatively low | Consumer acceptance and regulatory approval for novel food proteins can be lengthy |
| Scalable in controlled bioreactors, enabling food production independent of agricultural land and weather |
What might you want to genetically engineer fungi to do and why?
1. Produce pharmaceuticals and high-value therapeutic compounds
Fungi are natural producers of bioactive secondary metabolites including antibiotics, immunosuppressants, and anticancer agents. Engineering can dramatically boost yields. For example, engineered Aspergillus niger has been made to produce secondary metabolites at titres up to 4,500 mg/L — far exceeding natural production levels. Yeast has been engineered to produce rare anticancer saponins (e.g., polyphyllin II) normally extracted from endangered medicinal plants, providing a sustainable, controllable alternative to plant harvesting.
Why? To secure reliable supply of life-saving drugs independent of wild harvesting or chemical synthesis, reduce costs, and enable discovery of novel compounds through combinatorial biosynthesis of fungal enzymes and pathways.
2. Convert waste into biofuels, bioplastics, and industrial chemicals
Filamentous fungi can break down lignocellulosic biomass and waste streams into valuable products. The ligninolytic fungus Phanerochaete chrysosporium can generate biofuels, bioplastics, and pharmaceuticals from agricultural waste. Engineered strains show even greater degradation efficiency and product yields.
Why? To address the global waste crisis (an estimated 181.5 billion tonnes of lignocellulosic biomass generated annually) while creating economic value and reducing greenhouse gas emissions from waste burning and landfilling.
3. Bioremediate environmental pollutants
Fungi can be engineered to degrade a wide range of pollutants including heavy metals, synthetic polymers, dyes, pesticides, polycyclic aromatic hydrocarbons, and microplastics. Fungal mycelium networks act like "biological sponges" trapping contaminants, and fungal enzymes (e.g., laccases, manganese peroxidases) break down complex pollutants.
Why? To close circular economy loops by transforming hazardous waste into benign or recoverable resources, and to address the microplastics crisis — fungi can be "trained" to digest plastics that would otherwise persist for centuries.
4. Engineer climate-resilient agricultural solutions
Fungi can be engineered as biofertilisers, biocontrol agents, and carbon sequestration tools. Their natural ability to adapt to extreme conditions can be harnessed to reduce chemical inputs in agriculture.
Why? To reduce reliance on synthetic fertilisers and pesticides, enhance soil carbon storage, and develop sustainable agricultural practices resilient to climate change.
5. Create novel biomaterials with tailored properties
Genetic engineering can program fungi to produce materials with specific characteristics — strength, flexibility, water resistance, colour, or texture — by selecting and breeding strains or introducing foreign biosynthetic pathways.
Why? To replace petroleum-based plastics and animal-derived materials with tunable, biodegradable, low-carbon alternatives for packaging, construction, fashion, and automotive industries.
6. Manufacture industrial enzymes at high yields
Fungi are already used for large-scale enzyme production (e.g., cellulases, proteases, amylases). Engineering can enhance secretion efficiency, thermal stability, and substrate specificity.
Why? Enzymes are critical for countless industrial processes — food processing, detergent manufacturing, textile processing, paper production, and biofuel generation. Improved fungal enzyme factories increase efficiency and lower costs.
7. Produce novel foods and flavours
Engineered yeasts and fungi can produce specific flavour compounds, amino acids, and food ingredients at industrial scale. Precision-fermented food proteins are emerging as ethical alternatives to animal-derived dairy, egg, and meat proteins.
Why? To address the environmental impact of animal agriculture, enable food production independent of land and climate, and meet growing global protein demand sustainably.
What are the advantages of doing synthetic biology in fungi as opposed to bacteria?
| Feature | Bacteria (E. coli) | Fungi (yeasts and filamentous fungi) |
|---|---|---|
| Post-translational modifications (PTMs) | Very limited — generally lack eukaryotic PTM machinery (glycosylation, phosphorylation, acetylation, disulfide bond formation) | Extensive PTM capabilities — can perform complex eukaryotic modifications essential for functional protein production |
| Secretion capability | Export systems less developed; many products remain intracellular, requiring costly cell lysis | Naturally high-level secretors — many filamentous fungi evolved powerful secretion systems as decomposers, exporting enzymes directly into the medium |
| Protein folding | Limited ability to correctly fold complex eukaryotic proteins; prone to inclusion body formation | Proper folding machinery for disulfide bonds and multi-domain eukaryotic proteins; can express functional human/plant proteins |
| Natural product biosynthesis | Not natural producers of most complex secondary metabolites | Natural producers of antibiotics, immunosuppressants, anticancer agents, etc. — possess innate pre-mRNA splicing systems and abundant biosynthetic precursors |
| Substrate versatility | Narrow substrate range; typically requires refined sugars | Can utilise diverse low-cost feedstocks (lignocellulose, agricultural waste, food processing residues) |
| Growth and production cost | Very fast growth (minutes), extremely low media costs; ideal for simple proteins | Moderate growth rates (hours), but can be fermented on very low-cost agricultural byproducts, balancing cost |
| PTM complexity | Cannot perform human-type glycosylation; therapeutic proteins may be non-functional or immunogenic | Capable of humanised glycosylation pathways via glycoengineering, producing functional therapeutic proteins |
| Intracellular vs. extracellular | Products often accumulate intracellularly, requiring harsh lysis and complex purification steps | Products secreted into medium, simplifying downstream processing and enabling continuous production |
| Genetic tools maturity | Extremely well-developed, decades of optimisation | Rapidly advancing — CRISPR-Cas9, Cas12a, promoter/terminator libraries, landing-pad platforms for modular integration now available for many species |
| Threat level | Human pathogens exist (e.g., E. coli pathogenic strains), but lab strains generally safe | Most industrial fungi are safe (Generally Recognised as Safe status for many species); no endotoxin issues |
Key superiority for complex protein production: Bacteria are excellent for simple, prokaryotic proteins produced intracellularly at low cost. However, for eukaryotic proteins requiring proper folding, glycosylation, and disulfide bonds — which include most therapeutic proteins, industrial enzymes produced for human applications, and food proteins — fungal systems are essential. Filamentous fungi in particular combine the low media costs of bacterial systems with the eukaryotic processing capabilities of higher organisms, all while secreting products to simplify purification.
Key superiority for natural product discovery and production: Unlike E. coli, which lacks native secondary metabolite pathways, filamentous fungi are evolutionarily optimised to produce diverse bioactive small molecules. Their innate biosynthetic gene clusters can be activated, modified, or transferred, making them superior chassis for producing pharmaceutical compounds that bacteria simply cannot make.
Conclusion: Choose bacteria for simple, fast, cheap production of prokaryotic or non-glycosylated proteins. Choose fungi — particularly filamentous fungi and yeasts — when products require eukaryotic post-translational modifications, need to be secreted for easy purification, or are complex natural products that fungi naturally know how to make.
Assignment Part 3: First DNA Twist Order
📅 HTGAA 2026: Individual Final Project Submission Checklist
📋 Milestone Checklist & Progress Tracking
- Submit the Official Google Form (Deadline: March 20)
- Finalize the text for Draft Aim 1
- Polish the Final Project Summary abstract
- Select preferred tracks for the HTGAA Industry Council
- Generate and paste the shared link to the DNA Design Folder (Benchling/Kernel)
- Complete Week 2 Homework (Part 3: DNA Design Challenge)
- Design at least one (1) insert sequence and save it into the shared folder
- Document the target backbone vector on the project website
📝 Submission Copy & Technical Drafts
🔬 1. Final Project Summary
Context: Copy and paste this text directly into the designated abstract section of the Google Form.
This project introduces the Prometheus Symbiont, a paradigm-shifting bio-hybrid entity designed to systematically overcome the operational lifespan bottleneck of living biological components within engineering matrices. By isolating photosynthetic thylakoid membranes from Synechococcus elongatus and integrating them onto functionalized carbon nanotube anodes, we establish a direct, high-efficiency biophotovoltaic conversion interface. The architecture features an on-board microfluidic directed evolution platform to drive continuous cellular self-healing alongside a genetically encoded, dual-mode Calcium Ion ($\text{Ca}^{2+}$) central control interface. Driven by this bio-digital bridge, a silicon master chip dynamically toggles soft-robotic actuators between energy accumulation (“Grow”) and photoprotective shading (“Defense”), ultimately achieving carbon-neutral, self-renewing, long-endurance technological autonomy.
🎯 2. Core Project Aim 1 Draft
Context: Copy and paste this text into the Specific Aims section of the Google Form.
Aim 1: Engineering and Characterization of the Dual-Mode $\text{Ca}^{2+}$ Bio-Digital Communication Interface. We will clone and optimize a genetically encoded calcium indicator (GCaMP6s) under the control of a cyanobacterial promoter ($P_{psbAI}$) within Synechococcus elongatus PCC 7942. We will quantitatively characterize the ratiometric fluorescence output velocity ($\Delta F/F_0$) under simulated photoinhibitory stress spikes ($0$ to $2000 \ \mu\text{mol photons m}{-2}\text{s}{-1}$) to validate threshold detection parameters required for closed-loop machine actuator responses.
🧬 3. Week 2 DNA Design Challenge & Website Specifications
Context: Publish this section on your individual project website documentation page to satisfy the Part 3 assignment requirement.
| Parameter | Technical Specifications |
|---|---|
| Shared Design Directory | HTGAA_2026_Prometheus_Symbiont_DNA (Benchling / Asimov Kernel) |
| Insert Sequence Name | NS1-PpsbAI-RBS-GCaMP6s-TrrnB |
| Gene of Interest (GOI) | GCaMP6s (Genetically Encoded Calcium Indicator optimized for stress state capturing) |
| Target Backbone Vector | pAM1579 (Standard cyanobacterial integration vector targeting Neutral Site 1 ($NS1$) via homologous recombination) |
| Assembly Methodology | High-fidelity Gibson Assembly (Linear insert designed with 40-bp overlapping terminal homology arms) |