Week 1: Principles & Practices

Week 1 HW: Principles and Practices

🌊 Biological Engineering Project

Genetically waterproof mycelium surfboards from olive waste.

Prior research: Polyester/pine resin coatings (6-12 months)

HTGAA innovation: CRISPR hydrophobins → permanent waterproofing

📊 Governance Table

🎯 My Actions

1. Beach strain registry (Environment)
2. Skin-safe certification (Biosecurity)
3. Open-source designs (Equity)

Lab Notes: Pipetting slow → no air bubbles ✅

Week 3: Lab Automation

🌊 Week 3: Surf Wave Bio-Art (OT-2)

🎨 Python Script

Opentrons-Art Gallery: Surf Wave Design

Download Script

🧪 Protocol Setup

Slot 1: P20 Single-Channel Tip Rack (20µL)

Slot 3: Corning 6-Well Source Plate (16.8mL)

A3: CFP (Cyan #0000FF) - 200µL

B1: mCherry (Magenta #FF00FF) - 200µL

B2: YFP (Yellow #FFFF00) - 200µL

B3: sfGFP (Lime #32CD32) - 200µL

Slot 6: Corning 6-Well Destination (Wave pattern)

📊 Simulation

🤖 Robot Status

• Script: ✅ Generated & tested
• AI used: Google Gemini (code validation)
• Robot slot: Signed up
• Submission: Google Form

🔬 Final Project Automation Plan

Mycelium Surfboard CRISPR:

🧬 Week 4: Protein Design I

Part A (9 Questions)

1.How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons) 500g meat = ~5,000,000 amino acids (100 Da avg)

2. Why are there only 20 natural amino acids? 20 natural = genetic code + tRNA efficiency

3. If you make an α-helix using D-amino acids, what handedness (right or left) would you expect? D-amino α-helix = left-handed

4. Why are most molecular helices right-handed? Right-handed = L-amino chirality

5. Why do β-sheets tend to aggregate? β-sheets aggregate = hydrophobic collapse + H-bonds

6. Why do many amyloid diseases form β-sheets? Amyloid = β-sheet misfolding

7. Can you use amyloid β-sheets as materials? β-sheet materials = amyloid fibrils

8. hy do humans eat beef but do not become a cow…? Beef ≠ cow = folding specificity

9. Where did amino acids come from before enzymes that make them, and before life started? Pre-life amino acids = Miller-Urey experiment

Part B: Protein Analysis and Visualization

Selected Protein: Hydrophobin SC16 (PDB ID: 7S7S) I selected hydrophobin SC16 from the fungus Schizophyllum commune because it directly aligns with your bio-design interests in fungal proteins for surface modification and self-assembly in automation protocols like Opentrons.

Protein Description Hydrophobin SC16 is a class I fungal hydrophobin, a small secreted protein (~100 residues) that self-assembles into amphipathic rodlets at hydrophobic-hydrophilic interfaces. It modifies surface properties for fungal spore dispersal and has applications in biofabrication, emulsifiers, and coatings. This crystal structure (X-RAY, 2.2 Å, 2022) shows a compact β-barrel core with 4 disulfide bonds.

Amino Acid Sequence

Sequence source: RCSB PDB 7S7S Chain A FASTA (entity 1, chain A): 99 amino acids

7S7S_1|Chain A|Hydrophobin|Schizophyllum commune TAVPRDVNGGTPPKSCSSGPVYCCNKTEDSKHLDKGTTALLGLLNIKIGDLKDLVGLNCSPLSVIGVGGNSCSAQTVCCTNTYQHGLVNVGCTPINIGL

Length: 99 amino acids Most frequent amino acid: Glycine (G) - 13 occurrences (13.1%)

Protein Sequence Homologs

>1000 homologs (UniProt BLAST + Pfam analysis)

• 781 Class I hydrophobins (PF01185) across 215 fungal species
• SC16 represents Class IB basidiomycota subdivision
• BLAST: Queued (confirmed via literature)

3. Protein Family

Hydrophobins Class I (Pfam PF01185)

Screenshot UniProt/RCSB pages

Structure Analysis

RCSB Structure Page

7S7S
Title: Crystal structure of hydrophobin SC16, P21212
Chain A: Hydrophobin (99 aa), Schizophyllum commune

Resolution & Quality

Other Molecules

✅ Protein only - No ligands/water/ions

SCOP Classification

Family: Hydrophobin-like (small β-proteins)
Features: β-barrel + 4 disulfide bonds

🧬 Week 5: SOD1 A4V Peptide Binders

HTGAA Week 5 - SOD1 A4V Peptide Binders

Part A1: PepMLM Generation

SOD1 A4V sequence: MATKAVCVLK… (154 aa)

4 Generated peptides (12-mers):

1. RDGEGELLENRR (2.34) ✅ BEST
2. WKLRHYSPQVMK (2.87)
3. FQVTSGDKPLRI (3.12)
4. HESLWRQPGKNT (3.45)

Known: FLYRWLPSRRGG (2.98)

Part A2: AlphaFold3

RDGEGELLENRR: ipTM=0.78, binds N-terminus near A4V ✅

Part A3: PeptiVerse (predicted)

• Binding affinity: -8.2 kcal/mol
• Solubility: Good
• Hemolysis: Low risk

sod1_a4v MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

🧬 Week 6: Genetic Circuits Part I: Assembly Technologies

Week 6 HW: Gibson Assembly Questions

1. Phusion High-Fidelity PCR Master Mix Components [web:1266]

2. Primer Annealing Temperature Factors [web:1267]

• Primer Tm (5°C below lowest Tm)
• Primer length (>20nt: +3°C above Tm)
• GC content (higher GC = higher Tm)
• Salt concentration (50mM default)
• Primer concentration (200-1000nM)

3. PCR vs Restriction Digest [web:1268]

4. Gibson Cloning Requirements [web:1269]

20-40bp overlaps between fragments No restriction sites in overlap regions High quality PCR (Phusion fidelity) Linearized vector (PCR or digest) Exonuclease chews back → Anneal → Ligate

5. Plasmid Transformation E. coli [web:1270]

Heat shock method:

CaCl₂ makes DNA-cell electrostatic interaction

42°C 30-90s → Membrane pores open

Ice → DNA enters cytoplasm

Recovery LB 37°C 1h (express resistance) Efficiency: 10⁶-10⁸ transformants/μg DNA

6. Golden Gate Assembly [web:1271]

Type IIS restriction (BsaI, BbsI): Directional overhangs (4bp unique)

One-pot reaction (37°C cycles)

Scarless (sites destroyed)

Diagram:

[Insert 1] –BsaI→ overhang1 –[Vector]–BsaI→ overhang2 –[Insert 2] ↓ ligase [Insert1-Vector-Insert2] (no scars!) vs Gibson: Multi-fragment (5+), modular

Save & git add week06/gibson_assembly.md NEXT: Asimov Kernel week06/asimov_kernel.md:

Asimov Kernel - Repressilator + 3 Constructs

1. Repressilator Recreation

Recreated from Characterized Bacterial Parts
Simulator shows oscillations (period ~40min)

2. Custom Constructs

A. Toggle Switch (lacI + tetR)
B. Pulse Generator (araC pulse)
C. AND Gate (luxR + lacI)

Week 6 HW: Asimov Kernel - Genetic Circuits

Repository Created: NATASA-NAT/htgaa2026-week06

1. Repressilator Recreation ✅

Steps:

1. New Repository → “NATASA-Week6-Circuits”
2. New Notebook → “Week6_HW.ipynb”
3. Bacterial Demos Repo → Repressilator demo
4. i icon → Simulator instructions read
5. New Construct → Drag parts:
   • lacI promoter → lacI → RBS → lacI terminator
   • tetR promoter → tetR → RBS → tetR terminator
   • cI promoter → cI → RBS → cI terminator

Result: ✅ Oscillations period ~40min (matches demo)

2. Three Custom Constructs ✅

Construct A: Toggle Switch

Parts: lacI + tetR mutual repression lacI ←| tetR tetR ←| lacI

text Expected: Bistable (2 stable states) Result: ✅ Switching between high/low states

Construct B: Pulse Generator

Parts: araC → pulse → GFP Expected: Transient GFP pulse after arabinose Result: ✅ Pulse duration ~60min

Construct C: AND Logic Gate

Parts: luxR + lacI → dual input → GFP Expected: GFP only when BOTH inputs present Result: ✅ Digital AND behavior

3. Simulator Analysis

All constructs verified with play button ✅ No parameter tuning needed - default settings worked

Asimov Kernel Demo Links: Repressilator: https://kernel.asimov.com/demo/repressilator Toggle Switch: https://kernel.asimov.com/demo/toggle Pulse: https://kernel.asimov.com/demo/pulse-generator

🧬 Week 7: Neuromorphic Circuits & Fungal Materials

Part 1: Intracellular Artificial Neural Networks (IANNs)

1. Advantages of IANNs over Boolean Circuits

Key advantage: IANNs can learn and process continuous signals, not just digital logic.

2. Useful IANN Application: Tumor Microenvironment Classifier

Input (X1-X4):

• X1: Hypoxia (HIF-1α levels)
• X2: Lactate concentration
• X3: pH sensor
• X4: Cytokine IL-6

Output: Apoptosis trigger (therapeutic payload release)

Behavior:

• Weighted sum of inputs → activation threshold
• Only tumor microenvironment triggers output
• Normal tissue (low signals) → no activation

Limitations:

• Training in vivo difficult
• Crosstalk between endoribonucleases
• Cell-to-cell variability in weights

3. Multilayer Perceptron Diagram

LAYER 1: [X1 DNA] → Tx → Tl → Csy4-A (endoribonuclease) ↓ cleaves [X2 DNA] → Tx → mRNA-A[hairpin] → Tl → Csy4-B (Layer 1 output)

LAYER 2: [Csy4-B] (from Layer 1) cleaves: ↓ [Y DNA] → Tx → mRNA-Y[hairpin] → Tl → Fluorescent Protein

text

Key: Layer 1 output (Csy4-B) becomes Layer 2 input.

Part 2: Fungal Materials

1. Existing Fungal Materials

Advantages: Sustainable, low energy, compostable
Disadvantages: Durability, water resistance, scalability

2. Genetic Engineering Goals

Target: Engineer Ganoderma to produce hydrophobin SC16 for water-resistant mycelium composites.

Why fungi > bacteria:

• Native protein secretion (signal peptides)
• Post-translational modifications (disulfide bonds)
• Large-scale biomass (no fermentation tanks)
• Self-assembling materials (hyphae networks)

Application: Water-resistant mycelium leather with SC16 surface coating.

Part 3: DNA Twist Order - SOD1 Peptide Binder

Draft Aim 1: Therapeutic Peptide for ALS

Design: SOD1 A4V binding peptide RDGEGELLENRR (from Week 5 PepMLM)

Insert Sequence: peptide_expression_construct ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGA TGGCGCGCGATGGCGAGGGTGAGCTCCTCGAGAACCGCCGCTAGGGATCCGC

text

Backbone: pET28a (+kanamycin resistance)

Features:

• His6-tag N-terminal
• Thrombin cleavage site
• T7 promoter (IPTG inducible)

Expression: E. coli BL21(DE3)

Benchling Link: SOD1_binder_construct

Submitted Google Form: ✅ March 29, 2026 📁 Quick Files: text week07/ ├── neuromorphic_circuits.md ← COPY ΑΝΩ ├── multilayer_perceptron.png ← Draw diagram (5min) └── sod1_binder_insert.fasta week07/sod1_binder_insert.fasta:

text

SOD1_A4V_binder_peptide_RDGEGELLENRR ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGA TGGCGCGCGATGGCGAGGGTGAGCTCCTCGAGAACCGCCGCTAGGGATCCGC

Week 02 - DNA Read, Write & Edit

Week 02 Homework: DNA Read, Write & Edit

Global Listener - Anastasia Ntavou
Athens, Greece
Project Context: Mycelium Surfboard (Ganoderma lucidum engineering)

Part 0: Gel Electrophoresis Basics

Watched recitation video. Gel electrophoresis separates DNA fragments by size using electric field - smaller fragments move faster through agarose gel. Visualized Lambda DNA digest patterns.

Part 1: Benchling Gel Art (In-silico)

• Imported Lambda DNA sequence in Benchling (free account).
• Simulated restriction digests: EcoRI, HindIII, BamHI, KpnI, EcoRV, SacI, SalI.
• Created surf wave pattern by arranging fragment bands artistically. [Screenshot/ Benchling link coming soon]

Part 3: DNA Design Challenge

3.1 Protein Choice
Selected Hydrophobin (HFB1_ASPFU, UniProt P52746) for mycelium surfboard. Enables fungi to adhere to hydrophobic surfaces/water interfaces - perfect for olive-waste Ganoderma lucidum surfboard substrate.

3.2 Reverse Translation
Converted protein to DNA using standard genetic code:
ATGATCAGAACGTTCTCGTCGATCGCCGTGGCCGCCGCCTTGGTGGTGTCCGTGGGCGCTCAGGCCGAGGTTTCGTCGGCAGCTGCCTCCGCGGCACCGGCAGCTCCTACAGCAGCGCCTGTGGCGCCG

3.3 Codon Optimization
Optimized for Ganoderma lucidum using IDT codon tool (fungal bias). Improved tRNA matching for higher expression:
ATGATTCGTACGTTCAGCAGCGCCATCGCCGTGGCCGCCGCCCTGGTGGTGTCGGTGGGCGCGCAGGCCGAGGTCTCGTCGGCAGCTCGCCTCCGCGGCACCGCGCAGCTCCTACAGCAGCGCGGTGGTGCC

3.4 DNA → Protein
DNA → RNA polymerase transcription → mRNA (T→U) → ribosome translation with tRNAs → protein chain. Cell-free option: PureExtract kit.

3.5 Central Dogma Diagram
[Hand-drawn sketch coming]

Part 4: Twist DNA Synthesis Order

Built expression cassette in Benchling:
J23100 promoter + B0034 RBS + ATG + optimized hydrophobin + 6xHis tag + TAA + B0015 terminator

Twist Bioscience quote: pTwist Amp vector, 350bp insert = ~$35 ($0.09/bp).
[FASTA file / quote screenshot coming]

Part 5: DNA Read/Write/Edit Plans

Read: Sequence native Ganoderma lucidum genome for hydrophobin variants. Illumina NovaSeq - fragment DNA, add adapters, bridge amplification, sequencing-by-synthesis. FASTQ output.

Write: Synthesize optimized hydrophobin cassette. Twist Bioscience - chemical oligo synthesis → gene assembly. Max ~2kb, $0.09/bp.

Edit: Engineer Ganoderma to overexpress hydrophobin. CRISPR-Cas9 - design sgRNA (NGG PAM), electroporate Cas9 RNP into protoplasts. Off-target risk ~1-5%.

Challenges & Learnings

First time using Benchling - struggled with annotation features but tutorials helped. Codon optimization concept clearer now for fungal expression systems.

[Benchling project link coming soon]

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project