Week 7 HW: IANNs & Fungal Materials

Week 7: IANNs & Fungal Materials

This week covers two advanced synthetic biology paradigms: Intracellular Artificial Neural Networks (IANNs) for complex logical decision making, and the engineering of macro-scale Fungal Materials.

Part 1: Intracellular Artificial Neural Networks (IANNs)

1. Advantages over Traditional Boolean Genetic Circuits

While traditional genetic circuits rely on rigid, binary Boolean logic (AND, OR, NOT), Intracellular Artificial Neural Networks (IANNs) can process analog (continuous) signals. They are fundamentally capable of:

• Weight Tuning: By adjusting ribosome binding site (RBS) strengths or promoter affinities, users can “weight” different inputs.
• Complex Pattern Recognition: IANNs can classify complex metabolic states combining multiple molecular markers that might otherwise be too noisy for sharp Boolean switches to handle effectively.
• Non-linear computation: Using cooperative binding or enzymatic thresholds, they can perform fuzzy logic and handle biological noise much more robustly.

2. Application for an IANN

Application Target: A therapeutic “Cancer Cell Classifier” circuit.

• Inputs ($X_1, X_2, X_3$): Intracellular concentrations of three different oncogenic microRNAs (e.g., miR-21, miR-155) or cancer-specific transcription factors.
• Output ($Y$): Production of a pro-apoptotic protein (such as Bax or Caspase-9).
• Behavior: The IANN receives the concentrations of the input markers. Instead of firing if only one crosses a threshold (which might cause a false positive in a healthy cell), the network computes a weighted sum of the markers. Only if the integrated score crosses the hidden layer’s activation threshold does it trigger the output, executing cell death selectively.
• Limitations: Maintaining multiple distinct plasmids or thick gene cassettes places a massive metabolic load on the cell. Furthermore, crosstalk between similar biological components in different layers can cause short-circuits.

3. Diagram of a Multilayer Perceptron IANN

Here is a conceptual diagram of a multilayer perceptron where Layer 1 outputs an endoribonuclease (like Csy4), which in turn regulates (cleaves) the mRNA of a fluorescent protein in Layer 2.

graph TD
    classDef input fill:#e1f5fe,stroke:#03a9f4,stroke-width:2px;
    classDef layer1 fill:#f3e5f5,stroke:#9c27b0,stroke-width:2px;
    classDef layer2 fill:#e8f5e9,stroke:#4caf50,stroke-width:2px;
    classDef output fill:#fff3e0,stroke:#ff9800,stroke-width:2px;

    Input1[Input Signal X1<br/>e.g., Inducer]:::input
    Input2[Input Signal X2<br/>e.g., Inducer]:::input

    subgraph Layer 1: Hidden Layer
    L1Tx[Transcription Factor]:::layer1
    L1TF[Csy4 Endoribonuclease]:::layer1
    end

    Input1 --> L1Tx
    Input2 --> L1Tx
    L1Tx --> L1TF

    subgraph Layer 2: Output Layer
    L2mRNA[Target mRNA<br/>with Csy4 cut site]:::layer2
    L2Fluor[Fluorescent Protein]:::layer2
    end

    L1TF -- Cleaves Repressive Site --> L2mRNA
    L2mRNA -- Translation --> L2Fluor:::output

Part 2: Fungal Materials

1. Existing Fungal Materials

Fungal materials are primarily made by growing mycelium (the root-like structure of mushrooms) on an agricultural substrate.

• Mycelium Packaging (e.g., Evocative Mushroom Packaging): Used to replace styrofoam in shipping.
• Mycelium Leather (e.g., Mylo, Reishi): A vegan, biodegradable alternative to animal leather used in fashion and upholstery.
• Acoustic / Structural Panels: Mycelium mixed with hemp or wood chips, baked into bricks for construction.
• Advantages: Fully biodegradable, carbon-negative or neutral, upcycles agricultural waste (like corn stalks), and requires very little water/energy to grow.
• Disadvantages: Lower tensile strength compared to synthetic petroleum plastics, water sensitivity (can degrade or mold if not properly sealed or baked), and difficulties in scaling consistent macro-structures.

2. Genetic Engineering of Fungi

Application: Genetically engineer Aspergillus niger or Trichoderma reesei to secrete enzymes capable of degrading microplastics (e.g., PETase for PET plastics) or “forever chemicals” (PFAS). As the mycelium network grows through contaminated soil or water, it acts as a living, expansive bio-filter.

Advantages over Bacteria:

1. Macroscopic Networks: Fungi naturally form massive physical networks (hyphae) that can penetrate deep into solid substrates, which bacteria cannot do as effectively.
2. Eukaryotic Machinery: Fungi are eukaryotes. They possess the endoplasmic reticulum and Golgi apparatus necessary for complex post-translational modifications (like glycosylation), which are often required for large, active enzymes to fold properly.
3. Protein Secretion Capacity: Fungi are natural champions at secreting massive amounts of digestive enzymes into their extracellular environment, scaling far better than E. coli for industrial protein production.

Part 3: First DNA Twist Order

DNA Design Challenge

Building on my final project regarding MS2 Phage L-Protein Mutants (from Week 5).

• Insert Sequence: Synthetically designed, codon-optimized MS2 L-protein sequence with stabilizing mutations at the N-terminus.
• Backbone Vector: pET-28a(+)
• Purpose: The insert is flanked by restriction sites (e.g., NcoI and XhoI) to be cloned into the pET-28a(+) backbone, allowing robust IPTG-inducible expression of the mutated L-protein in E. coli BL21(DE3) cells for subsequent stability assays. The presence of the vector’s N-terminal His-tag will allow for easy IMAC purification.