🧬 Week 9: Cell-Free Systems
Global Listener — 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 — 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:
- Membrane proteins — toxic to cells; cell-free allows expression in detergent/lipid environments
- Rapid prototyping — screening dozens of protein variants in hours without transformation cycles
2. Components of a cell-free expression system:
| Component | Role |
|---|---|
| Cell extract (ribosomes, polymerases, tRNAs) | Core transcription/translation machinery |
| DNA template | Encodes the protein of interest |
| NTPs | Building blocks for RNA synthesis |
| Amino acids | Building blocks for protein synthesis |
| Energy source (ATP, creatine phosphate) | Powers translation |
| Mg²⁺, K⁺ salts | Stabilize ribosomes and enzymes |
| RNase inhibitor | Protects mRNA from degradation |
3. Energy provision in cell-free systems:
Creatine phosphate + creatine kinase system: creatine phosphate donates phosphate to ADP → ATP. Sustains translation for 2–4 hours. Alternative: phosphoenolpyruvate (PEP) system.
4. Prokaryotic vs. eukaryotic cell-free:
| Feature | Prokaryotic (E. coli) | Eukaryotic (wheat germ) |
|---|---|---|
| Cost | Low | Higher |
| PTMs | Limited | Glycosylation, disulfide bonds |
| Best for | Simple cytosolic proteins | Complex eukaryotic proteins |
- Prokaryotic: Express LAC2 laccase for initial activity testing — fast, cheap
- Eukaryotic: Express hydrophobin SC16 — requires 4 disulfide bonds; eukaryotic system provides correct oxidizing environment
5. Optimizing cell-free expression of a membrane protein:
- Add detergents (DDM) or liposomes to mimic membrane environment
- Use lipid nanodiscs to solubilize protein as it’s synthesized
- Lower temperature (25°C) for slower, better-folded synthesis
- Add chaperones (GroEL/GroES) to the extract
6. Troubleshooting low protein yield:
| Reason | Troubleshooting |
|---|---|
| mRNA degradation | Add RNase inhibitor; use circular template |
| Rare codons | Codon-optimize for extract organism |
| ATP depletion | Increase creatine phosphate; fed-batch approach |
Kate Adamala: Synthetic Minimal Cell Design
Function: Hydrophobin-secreting synthetic cell for mycelium surface waterproofing
1. Function:
- What it does: Produces and secretes hydrophobin SC16 that self-assembles on G. lucidum hyphal surfaces
- Input: Glucose (energy) + DNA template encoding SC16
- Output: Secreted SC16 that coats nearby mycelium surfaces → WCA > 120°
- Cell-free alone? No — encapsulation needed to concentrate protein near mycelium surface
- GMO natural cell? Yes — this is our main project approach; synthetic cell could serve as protein delivery vehicle alongside
2. Components:
- Membrane: POPC phospholipids + cholesterol (4:1 ratio)
- Encapsulate: E. coli cell-free Tx/Tl + SC16 gene under T7 promoter + ATP regeneration (creatine phosphate + creatine kinase) + DsbC oxidase (for disulfide bonds)
- Tx/Tl system: E. coli extract — sufficient with added oxidase for SC16 disulfide bonds
- Communication: OmpF porin (UniProt P02931) in membrane — allows glucose import and SC16 export
3. Experimental details:
- Lipids: POPC (Avanti #850457), cholesterol (Sigma C8667)
- Genes: SC16 hydrophobin (codon-optimized), OmpF porin, T7 RNA polymerase
- Measurement: Water contact angle (sessile drop) on mycelium-coated glass slide
Part B: Final Project Connection
Cell-free systems enable rapid SC16 validation before committing to slow G. lucidum transformation:
- Express His-tagged SC16 in PURExpress (NEB)
- Verify production by SDS-PAGE + anti-His western blot
- Test self-assembly by water contact angle on glass slide
- If cell-free SC16 achieves WCA > 120° → confirms design before in vivo work
Peter Nguyen: Cell-Free Systems in Materials
Application field: Textiles/Fashion
One-sentence pitch: Freeze-dried cell-free systems embedded in athletic wear that detect and respond to lactic acid buildup during exercise.
How it works: A biosensor construct encoding a lactic acid-responsive transcription factor (LldR) is freeze-dried into textile fibers. When the athlete sweats, water rehydrates the cell-free reaction. LldR detects lactate → activates GFP or a colorimetric reporter → fabric changes color when athlete reaches anaerobic threshold. The reaction is self-contained, single-use, and requires no external power source.
Societal challenge addressed: Overtraining and lactic acid accumulation cause muscle fatigue and injury in athletes. Real-time, non-invasive lactate monitoring embedded in clothing could prevent injury and optimize performance without wearable electronics.
Addressing cell-free limitations:
Activation: Sweat provides sufficient water for rehydration
Stability: Freeze-drying at -80°C preserves activity for
12 months at room temperature (demonstrated in literature)
One-time use: Each patch is single-use; patches can be integrated as disposable inserts in garment panels
Ally Huang: Genes in Space — Mock Proposal
Using BioBits® cell-free protein expression system
Background (max 100 words)
Long-duration spaceflight causes significant muscle atrophy in astronauts due to microgravity-induced changes in protein synthesis. Current monitoring requires blood draws and laboratory equipment unavailable on spacecraft. A lightweight, freeze-dried biosensor using cell-free protein synthesis could enable real-time, non-invasive monitoring of muscle health biomarkers in space, where resources are severely constrained. This is significant for humanity as it directly enables longer and safer deep-space missions.
Molecular target (max 30 words)
Myostatin (GDF-8) — a protein that inhibits muscle growth. Elevated myostatin levels indicate muscle atrophy progression in microgravity conditions.
Relevance to space biology challenge (max 100 words)
Myostatin is upregulated in microgravity, directly causing muscle wasting in astronauts. A cell-free biosensor detecting myostatin in urine or saliva would provide a non-invasive, equipment-free readout of muscle atrophy progression. Unlike blood-based assays, this approach requires no centrifuge or trained personnel — critical constraints in space. The freeze-dried format means the biosensor survives launch conditions and long storage without refrigeration.
Hypothesis (max 150 words)
If a cell-free biosensor encoding a myostatin-responsive genetic circuit (myostatin aptamer → toehold switch → GFP reporter) is freeze-dried and rehydrated with astronaut saliva, it will produce detectable fluorescence proportional to myostatin concentration. We hypothesize that myostatin levels >1 ng/mL (indicative of early atrophy) will activate GFP expression detectable with the P51 Molecular Fluorescence Viewer within 2 hours of rehydration. This would provide a simple, portable, and reagent-minimal method for weekly muscle health monitoring aboard the ISS or future deep-space missions.
Experimental plan (max 100 words)
- Sample: Astronaut saliva (collected weekly)
- Controls: Known myostatin concentrations (0, 0.5, 1, 5 ng/mL)
- Protocol: Rehydrate freeze-dried BioBits® reaction with 10µL saliva → incubate 37°C 2h using miniPCR® thermal cycler → measure fluorescence with P51 Viewer
- Data collected: Fluorescence intensity vs myostatin concentration standard curve
- Expected outcome: Linear fluorescence response enabling quantitative myostatin monitoring