Week 9 — Cell-Free Systems

PART 1

Question 1: What are the main advantages of cell-free protein synthesis (CFPS) regarding flexibility and control, and name two cases where it is more beneficial?

Since it is an open system, you have direct control over the chemical environment (pH, redox potential, and salts) and can add synthetic components like non-natural amino acids without being restricted by a cell membrane.

Case 1: Production of cytotoxic proteins that would otherwise kill a living host cell. Case 2: Efficient incorporation of labeled isotopes or synthetic tags for precise protein engineering.

Question 2: What are the main components of a cell-free expression system and what is the role of each?

  • Cell Extract (Lysate): Provides the biological machinery (ribosomes, tRNAs, and translation factors).
  • Energy Substrates (NTPs): Supplies the chemical fuel (ATP and GTP) to power the synthesis.
  • Amino Acids: The building blocks used to assemble the protein chain.
  • DNA/RNA Template: The genetic instructions for the specific target protein.
  • Salts and Buffers: Maintain pH and provide essential ions like $Mg^{2+}$ and $K^{+}$ for enzyme activity.

Question 3: Why is energy provision regeneration critical, and what method would you use to ensure a continuous ATP supply?

Protein synthesis is energy-intensive and produces inorganic phosphate as a byproduct, which inhibits the reaction. Without regeneration, the system runs out of fuel and stops within minutes.

Method: The Creatine Phosphate / Creatine Kinase system. The enzyme (kinase) transfers a phosphate group from creatine phosphate back to ADP, continuously recycling it into ATP.

Question 4: Compare prokaryotic versus eukaryotic cell-free systems. Choose a protein for each and explain why.

Prokaryotic (E. coli): Fast, high-yield, and inexpensive. I would choose T7 RNA Polymerase, as it is a bacterial protein that functions perfectly without complex folding or sugars.

Eukaryotic (e.g., Wheat Germ or CHO): Slower and more expensive, but capable of complex folding. I would choose Human Antibodies, because they require specific disulfide bonds and glycosylation (sugars) that only eukaryotic systems can provide.

Question 5: How would you design an experiment for a membrane protein, and what challenges would you address?*

Design: Incorporate artificial hydrophobic environments like nanodiscs or liposomes directly into the reaction mix.

Challenges: Membrane proteins are hydrophobic and tend to aggregate (clump together) in water-based buffers.

Solution: Use co-translational insertion, where the protein embeds itself into the added lipids as it is being synthesized by the ribosome, keeping it stable and functional.

Question 6: Describe three possible reasons for low yield and suggest a troubleshooting strategy for each.

Magnesium Imbalance: Incorrect Mg levels stop ribosome function. Strategy: Perform a magnesium titration to find the optimal concentration.

Template Degradation: Nucleases in the extract break down the DNA/RNA. Strategy: Add RNase inhibitors or protective proteins like GamS to shield the template.

Codon Bias: The gene uses instructions that are “rare” for the extract’s machinery. Strategy: Perform codon optimization on the DNA sequence to match the tRNA abundance of the lysate.

PART 2

    1. Pick a function and describe it

    • Function: synthetic cell that detects lactate and produces fluorescence

    • a. What would your synthetic cell do What is the input and what is the output

      • Input: lactate
      • Output: GFP

    • b. Could this function be realized by cell free Tx Tl alone without encapsulation

      • Partially yes, but with low stability and no environmental control

    • c. Could this function be realized by genetically modified natural cell

      • Yes, easier in bacteria such as E coli

    • d. Describe the desired outcome of your synthetic cell operation

      • Detect lactate and generate a proportional fluorescence response
    1. Design all components that would need to be part of your synthetic cell

    • a. What would be the membrane made of

      • Liposomes made of POPC and cholesterol

    • b. What would you encapsulate inside Enzymes small molecules

      • Bacterial Tx Tl system
      • DNA with lactate responsive circuit
      • GFP
      • LDH
      • NAD+

    • c. Which organism your Tx Tl system will come from

      • E coli based system for simplicity

    • d. How will your synthetic cell communicate with the environment

      • Passive diffusion of lactate or transport via LldP
    1. Experimental details

    • a. List all lipids and genes

      • Lipids

        • POPC
        • cholesterol

      • Genes

        • lldR
        • lldPRD
        • gfp
        • ldhA
        • lldP

    • b. How will you measure the function of your system

      • GFP fluorescence
      • Lactate consumption assay
      • Pyruvate measurement