Week 09 HW: Cell-Free Systems

1. Advantages of CFPS

The main advantage is that CFPS is an open system. Unlike in vivo methods, there is no cell membrane, allowing direct access to the reaction.

  • Flexibility: You can adjust $Mg^{2+}$ levels, add chaperones, or use non-natural amino acids easily.
  • Toxic Proteins: You can produce proteins that would normally kill a living host cell.
  • Speed: It enables “benchtop” production in hours rather than days of cell culture.

2. Main Components

  • Cell Extract (Lysate): The “machinery” (ribosomes, tRNAs, enzymes).
  • DNA Template: The “instructions” for the protein.
  • Energy System: ATP/GTP and a regeneration substrate (e.g., PEP).
  • Amino Acids: The “building blocks.”
  • Salts/Cofactors: Specifically $Mg^{2+}$ and $K^{+}$ for ribosome function.

3. Energy Provision

Why it’s critical: Protein synthesis is energy-expensive. Without a regeneration system, ATP is depleted in minutes by side reactions, stopping production.

  • Method: Use Creatine Phosphate and Creatine Kinase. This pair re-phosphorylates ADP back into ATP continuously during the reaction.

4. System Comparison: Prokaryotic vs. Eukaryotic

SystemHost ExampleAdvantagesBest For
ProkaryoticE. coliHigh yield, fast, cost-effective.Simple proteins (e.g., GFP).
EukaryoticWheat GermComplex folding, post-translational mods.Human proteins (e.g., EPO).

5. Membrane Protein Design

Challenge: These proteins are hydrophobic and aggregate in water-based lysates. Solution: Add Nanodiscs or Liposomes to the reaction. These provide a lipid scaffold for the protein to insert into as it is being built.

6. Troubleshooting Low Yields

  1. Template Degradation: Add RNase inhibitors to protect the mRNA/DNA.
  2. Magnesium Levels: Perform a $Mg^{2+}$ titration; ribosomes are extremely sensitive to salt concentrations.
  3. Codon Bias: Use a specialized lysate supplemented with rare tRNAs if the gene source and lysate source are different organisms.

Part 2: Synthetic Minimal Cell Design

An encapsulated arsenic biosensor for water quality monitoring.

  • Chassis-> E. coli-based Tx/Tl system encapsulated in a POPC/POPG phospholipid membrane.
  • Communication-> Alpha-hemolysin (αHL) pores allow arsenic ions to permeate the membrane.
  • Genetic Circuit-> An arsR repressor linked to a GFP reporter gene.
  • Mechanism-> Arsenic binding to ArsR triggers the expression of GFP.
  • Safety-> The system is non-replicative and biocontained, making it safe for environmental deployment.

Part 3: BioBits® in Space - Muscle Atrophy Diagnostic

Monitoring astronaut health via freeze-dried synthetic biology.

  • Challenge: Microgravity-induced muscle atrophy using the Myostatin (MSTN) gene sequence, a regulator of muscle growth.
  • Hypothesis: Freeze-dried BioBits® pellets can be rehydrated with astronaut samples to provide a visible fluorescent readout of MSTN levels.
  • Implementation Plan:
    1. Deploy freeze-dried BioBits® containing an MSTN-responsive circuit.
    2. Rehydrate with sample DNA/RNA.
    3. Quantify results using the P51 Fluorescence Viewer.
    4. Utilize non-target DNA as a negative control to ensure specificity.