Homework

Weekly homework submissions:

  • 🧬 Week 1: Principles & Practices

    🌊 Biological Engineering Project Genetically waterproof mycelium surfboards from olive waste. Prior research: Polyester/pine resin coatings (6-12 months) HTGAA innovation: CRISPR hydrophobins β†’ permanent waterproofing πŸ“Š Governance Table Criteria Option 1 Option 2 Option 3 Biosecurity βœ“ βœ“ βœ“ Lab Safety βœ“ βœ“ βœ“ Environment βœ“ βœ“ βœ“ Governance Options Option 1: Regulatory Notification Requirement Purpose: Currently no specific EU regulation targets mycelium GMM composites for consumer products. Propose mandatory notification to national authority (Hellenic Ministry of Rural Development) before production begins. Design: Manufacturer submits safety dossier; authority reviews within 90 days. Assumptions: Assumes regulatory capacity exists; may underestimate review backlog. Risks: Overregulation could stifle innovation; under-review could miss risks.

  • 🧬 Week 02 - 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. Benchling project: View sequence Part 3: DNA Design Challenge 3.1 Protein Choice Selected Hydrophobin SC16 (Schizophyllum commune, UniProt D8QCG9, PDB 7S7S) for mycelium surfboard. Class I hydrophobin that self-assembles into amphipathic rodlet films at hydrophobic interfaces β€” ideal for waterproofing Ganoderma lucidum mycelium composites.

  • 🌊 Week 3: Lab Automation

    🎨 Python Script Download Script **Opentrons-Art Gallery: ** Surf Wave Design πŸ§ͺ Protocol Setup Slot 1: P20 Single-Channel Tip Rack (20Β΅L) Slot 3: Corning 6-Well Source Plate (16.8mL) A3: CFP (Cyan #0000FF) - 200Β΅L B1: mCherry (Magenta #FF00FF) - 200Β΅L B2: YFP (Yellow #FFFF00) - 200Β΅L

  • 🧬 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) Why are there only 20 natural amino acids? 20 natural = genetic code + tRNA efficiency If you make an Ξ±-helix using D-amino acids, what handedness (right or left) would you expect? D-amino Ξ±-helix = left-handed

  • 🧬 Week 5: SOD1 A4V Peptide Binders

    Part A1: PepMLM Generation SOD1 A4V sequence (154 aa): MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTS AGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVV HEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ A4V mutation: Alanine β†’ Valine at position 4 Generated peptides (12-mers) via PepMLM-650M: Rank Peptide Perplexity Notes 1 RDGEGELLENRR 2.34 βœ… BEST β€” lowest perplexity 2 WKLRHYSPQVMK 2.87 Good candidate 3 FQVTSGDKPLRI 3.12 Moderate 4 HESLWRQPGKNT 3.45 Weakest of generated Known FLYRWLPSRRGG 2.98 Reference binder Lower perplexity = higher model confidence in binding

  • 🧬 Week 6: Genetic Circuits Part I: Assembly Technologies

    1. Phusion High-Fidelity PCR Master Mix Components Component Purpose Phusion DNA Polymerase High fidelity (52x Taq), fast extension dNTPs DNA building blocks MgClβ‚‚ Polymerase cofactor (NHβ‚„)β‚‚SOβ‚„ Stabilizes polymerase Betaine GC-rich templates DMSO Reduces secondary structure 2. Primer Annealing Temperature Factors Primer Tm (5Β°C below lowest Tm) Primer length (>20nt: +3Β°C above Tm) GC content (higher GC = higher Tm) Salt concentration (50mM default) Primer concentration (200-1000nM) 3. PCR vs Restriction Digest Feature PCR Restriction Digest Linear fragments Primers define ends Restriction sites Protocol 30 cycles (denature/anneal/extend) 1-2h 37Β°C digestion Advantages Scarless, any sequence Fast, cheap Gibson use Overlap primers (20-40bp) Compatible overhangs 4. Gibson Cloning Requirements 20-40bp overlaps between fragments No restriction sites in overlap regions High quality PCR (Phusion fidelity) Linearized vector (PCR or digest) Exonuclease chews back β†’ Anneal β†’ Ligate

  • 🧬 Week 7: Neuromorphic Circuits & Fungal Materials

    Part 1: Intracellular Artificial Neural Networks (IANNs) 1. Advantages of IANNs over Boolean Circuits Feature Boolean Circuits IANNs Logic ON/OFF only Analog weights Complexity n inputs = 2ⁿ truth table Continuous functions Learning Fixed Trainable weights Example AND/OR gates Pattern recognition Key advantage: IANNs can learn and process continuous signals, not just digital logic.

  • 🧬 Week 9: Cell-Free Systems

    Global Listener β€” Anastasia Ntavou | Athens, Greece Project: Mycelium Surfboard (Ganoderma lucidum engineering) Part A: General Questions 1. Advantages of cell-free protein synthesis: Cell-free systems bypass living cells, offering: Flexibility: Any DNA template added directly β€” no transformation needed. Toxic proteins expressible freely. Control: Reaction conditions (pH, redox, cofactors) tunable without affecting cell viability. Two cases where cell-free beats in vivo:

  • 🧬 Week 10: Advanced Imaging & Measurement

    Global Listener β€” Anastasia Ntavou | Athens, Greece Project: Mycelium Surfboard (Ganoderma lucidum engineering) Final Project Measurement Plan 1. SC16 Hydrophobin Expression Verification What: Presence and molecular weight of SC16 (~10 kDa) Method: SDS-PAGE + anti-His western blot Alternative: MALDI-TOF MS β€” confirms exact MW and detects truncations 2. Water Contact Angle (WCA) What: Surface hydrophobicity of engineered vs. wild-type mycelium Method: Sessile drop goniometry β€” 5Β΅L drop on mycelium-coated glass Target: > 120Β° (engineered) vs. < 30Β° (wild-type) 3. Disulfide Bond Verification What: Correct SC16 folding (4 disulfide bonds essential) Method: Non-reducing SDS-PAGE + MS peptide mapping 4. Composite Mechanical Properties What: Flexural strength, compressive strength, modulus Method: ASTM D790 (3-point bend), ASTM D695 (compression) 5. Compostability What: Mass loss over 28 days Method: ASTM D5338 at 58Β°C / 60% humidity Target: > 90% mass loss Waters Mass Spectrometry Questions Part I β€” eGFP Molecular Weight Using ExPASy ProtParam on the provided sequence (239 aa with His-tag):

  • 🧬 Week 11: Bioproduction & Cloud Labs

    Global Listener β€” Anastasia Ntavou | Athens, Greece Project: Mycelium Surfboard (Ganoderma lucidum engineering) Part A: 1,536 Pixel Collective Artwork β€” Olive Wave View Olive Wave submission Concept: A stylized wave in olive-green and ocean-blue fluorescent proteins β€” representing Greek olive agriculture meeting the ocean, the two ecosystems at the heart of this project. Fluorescent proteins: sfGFP (olive green), mTurquoise2 (wave foam blue), mCherry (deep background) Pattern: Wave crest in mTurquoise2, body in sfGFP, background in mCherry Part B: Cell-Free Protein Synthesis β€” SC16 Hydrophobin| Selected protein: Hydrophobin SC16 (directly relevant to final project)

  • 🧬 Week 12: Building Genomes

    Global Listener β€” Anastasia Ntavou | Athens, Greece Project: Mycelium Surfboard (Ganoderma lucidum engineering) Reflection: Genome-Scale Engineering & the Mycelium Surfboard This week’s lectures on synthetic genomes β€” from JCVI-syn3.0 (473 genes) to Sc2.0 β€” provided important context for the mycelium surfboard project. Minimal genomes: JCVI-syn3.0 has ~30% genes with unknown function. For G. lucidum (~49 Mb, ~16,000 genes), the complexity is vastly greater β€” reinforcing why targeted CRISPR knock-in (2 genes) is the right strategy rather than whole-genome redesign.

  • 🧬 Week 13: Biodesign & Engineered Living Materials

    Global Listener β€” Anastasia Ntavou | Athens, Greece Final Project Work Documentation Connection to Engineered Living Materials This week’s theme β€” engineered living materials (ELMs) β€” is the conceptual heart of the mycelium surfboard project. The surfboard is a living material: mycelium grows, self-organizes, and produces hydrophobin SC16 that permanently modifies its surface. What makes this an ELM:

  • 🧬 Week 14: Bio Design & Bio Fabrication

    Global Listener β€” Anastasia Ntavou | Athens, Greece Final Project Completed Reflection: Bio Design & Bio Fabrication Suzanne Lee (Biocouture) and Christina Agapakis (Ginkgo Bioworks) represent the frontier this project aims toward: biology as a design material, not just a research tool. Bio Design: The surfboard is not just an engineering project β€” it is a design object. The choice of olive pomace ties the product to Greek landscape and identity. A Cretan olive grove becomes part of the surfboard’s material story.