🧬 Week 11: Bioproduction & Cloud Labs
Global Listener — Anastasia Ntavou | Athens, Greece Project: Mycelium Surfboard (Ganoderma lucidum engineering)
Part A: 1,536 Pixel Collective Artwork — Olive Wave
Concept: A stylized wave in olive-green and ocean-blue fluorescent proteins — 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 — SC16 Hydrophobin|
Selected protein: Hydrophobin SC16 (directly relevant to final project)
Expression plan:
- Template: Linear PCR product with T7 promoter — SC16 CDS — His6 tag — T7 terminator
- Extract: PURExpress (NEB) — reconstituted E. coli system
- Modification: Replace DTT with 0.5mM oxidized glutathione (GSSG) to enable SC16 disulfide bond formation
- Incubation: 37°C, 2 hours
- Verification: Anti-His western blot; water contact angle on coated glass
Why cell-free for SC16? Validates protein function in hours before investing weeks in fungal transformation. Cell-Free Reaction Components
| Component | Role |
|---|---|
| E. coli Lysate (BL21 DE3 Star) | Contains ribosomes, RNA polymerase, tRNAs, and all translation machinery needed for protein synthesis |
| Potassium Glutamate | Maintains ionic strength and stabilizes ribosomes |
| HEPES-KOH pH 7.5 | Buffer — maintains stable pH for enzymatic reactions |
| Magnesium Glutamate | Mg²⁺ cofactor essential for ribosome function and polymerase activity |
| Potassium phosphate (mono/dibasic) | Energy regeneration buffer; maintains phosphate pool |
| Ribose + Glucose | Carbon energy sources for ATP regeneration |
| AMP, CMP, GMP, UMP | Nucleotide building blocks for RNA synthesis (transcription) |
| Guanine | Purine base — converted to GTP via salvage pathway for transcription |
| 17 Amino Acid Mix + Tyr + Cys | Building blocks for translation; Tyr and Cys added separately due to solubility |
| Nicotinamide | NAD⁺ precursor — supports redox reactions in energy metabolism |
| Nuclease Free Water | Backfill to final reaction volume; prevents RNA/DNA degradation |
1-hour PEP-NTP vs 20-hour NMP-Ribose-Glucose master mix
The 1-hour PEP-NTP mix uses phosphoenolpyruvate (PEP) as a fast energy source and pre-formed NTPs for immediate transcription — optimized for short, high-yield reactions. The 20-hour NMP-Ribose-Glucose mix uses nucleoside monophosphates and sugars that are metabolically converted to NTPs, sustaining lower-level expression over a longer period. The extended mix trades peak yield for longevity, making it suitable for slow-folding or complex proteins like hydrophobins.
Bonus: Guanine is converted to GMP via the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) salvage pathway present in the E. coli lysate, allowing GTP synthesis without direct GMP addition.
Part C: Global Experiment Master Mix Design
Goal: Optimize SC16 cell-free expression
| Variable | Condition A | Condition B | Condition C |
|---|---|---|---|
| Mg²⁺ | 4 mM | 6 mM | 8 mM |
| Template | 5 nM | 10 nM | 20 nM |
| Redox | DTT 1mM | GSSG 0.5mM | None |
| Temperature | 25°C | 30°C | 37°C |
Readout: Anti-His western band intensity + water contact angle measurement
Cloud lab advantage: All 12 conditions run simultaneously with automated liquid handling — impossible by hand in a single day. Fluorescent Protein Biophysical Properties
| Protein | Key property affecting cell-free expression |
|---|---|
| sfGFP | Fast maturation (~30 min); oxygen-dependent chromophore formation; robust folding — ideal reference protein for cell-free |
| mRFP1 | Slow maturation (~4h); prone to aggregation at high concentrations; less bright than newer RFPs |
| mKO2 | Orange fluorescent; requires oxygen for chromophore maturation; moderate maturation time ~1h |
| mTurquoise2 | Cyan fluorescent; fast maturation; high quantum yield; sensitive to acidic pH — may lose fluorescence below pH 6 |
| mScarlet_I | Bright red; fast maturation (~1h); monomeric — reduces aggregation risk in cell-free |
| Electra2 | Near-infrared fluorescent; requires biliverdin chromophore (not auto-catalytic) — may need exogenous biliverdin addition to cell-free reaction |
Hypothesis for improving fluorescence
Protein: mTurquoise2
Reagent: Increase HEPES-KOH buffer concentration to maintain pH > 7.0
Expected effect: mTurquoise2 is acid-sensitive — maintaining neutral-to-basic pH throughout the 36-hour incubation will prevent chromophore protonation and preserve fluorescence signal over the full reaction period.
Part D (Optional): Build-A-Cloud-Lab — MycoCloud
Concept: Distributed mycelium composite testing platform
| Module | Function |
|---|---|
| OT-2 substrate robot | Automated olive pomace + hemp mixing at precise ratios |
| Inoculation station | Sterile liquid spawn at 10% w/w |
| Environmental chamber | 28°C / 90% RH with CO₂ monitoring |
| Mini mechanical tester | 3-point bend on 1cm³ samples |
| WCA station | Automated sessile drop goniometer |
Why distributed? Nodes worldwide could run substrate experiments with local waste (olive pomace in Greece, grape marc in France, hemp in Netherlands) — enabling global comparative mycelium composite research.