Week 9 HW: Cell free systems

Homework Part A: General and Lecturer-Specific Questions

General homework questions

Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production.

In traditional in vivo (inside the cell) methods, the cell’s primary goal is its own survival. In CFPS, the goal is purely production.

Experimental Control: You can directly manipulate the environment. You can add non-natural amino acids, adjust redox potential, or add chaperones at precise concentrations without the cell’s homeostatic mechanisms fighting back.
Flexibility: There is no “transformation” step. You can add linear DNA or mRNA directly to the mix, enabling rapid prototyping (Design-Build-Test cycles).

Two Cases where CFPS is superior:

Cytotoxic Proteins: Producing proteins that would kill a living host (e.g., antimicrobial peptides or certain toxins) is much easier in vitro because the system doesn’t need to stay “alive.”
Incorporation of Non-Canonical Amino Acids (ncAAs): CFPS allows for the site-specific insertion of synthetic amino acids to create proteins with new chemical properties, which is often difficult in cells due to transport issues or toxicity.

Describe the main components of a cell-free expression system and explain the role of each component.

Cell Extract (Crude Lysate): Provides the core machinery: Ribosomes, aminoacyl-tRNA synthetases, and initiation/elongation factors.
Cell-free protein synthesis template DNA or mRNA: Encodes the protein of interest.
Energy Source High-energy molecules (e.g., Phosphoenolpyruvate or Creatine Phosphate): to fuel the reaction.
Amino Acids: The raw building blocks for the protein chain.
Nucleotides (NTPs): Required for transcription (if starting from DNA) and energy transfer.
Salts and Buffers (e.g., Magnesium and Potassium): To maintain pH and stabilize the folding of the machinery.

Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.

Protein synthesis is energetically expensive. Every peptide bond requires the hydrolysis of multiple ATP and GTP molecules. If the energy supply is exhausted, translation stops, leading to low yields. One common method is the Secondary Energy Solution using Creatine Phosphate and Creatine Kinase. The kinase enzyme transfers a phosphate group from creatine phosphate to ADP, constantly “recharging” the ATP pool within the tube.

Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

Prokaryotic (e.g., E. coli): High yield, fast, and inexpensive. However, it lacks post-translational modification (PTM) capabilities.

Protein to produce: Insulin. While complex, it is a small protein that can be efficiently produced and folded in optimized E. coli systems.

Eukaryotic (e.g., CHO or Wheat Germ): Lower yield but capable of complex folding and PTMs like glycosylation.

Protein to produce: Monoclonal Antibodies. These require complex disulfide bond formation and glycosylation to be functional, which prokaryotic systems cannot easily handle.

How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup.

Membrane proteins are notoriously difficult because they are hydrophobic and require a lipid environment to fold correctly; otherwise, they aggregate and become useless.

Challenges and Solutions:

Challenge: Lack of a lipid bilayer.
Solution: Supplement the CFPS reaction with nanodiscs or synthetic liposomes. These provide a “landing pad” for the protein to insert itself into as it is being synthesized.
Challenge: Detergent toxicity.
Solution: Use detergent-free CFPS or mild surfactants that stabilize the protein without denaturing the translation machinery.

Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.

Template Degradation: Check the purity of your DNA/mRNA. Use RNase inhibitors to prevent the degradation of the blueprint during the reaction.
Energy Depletion: Analyze the duration of the reaction. If it stops prematurely, increase the concentration of the secondary energy source or implement a dialysis system to remove byproduct phosphate.
Poor Protein Folding: If the protein is being made but is insoluble (forming pellets), try lowering the incubation temperature or adding molecular chaperones (like DnaK/J) to the extract.

Homework question from Peter Nguyen

Freeze-dried cell-free systems can be incorporated into all kinds of materials as biological sensors or as inducible enzymes to modify the material itself or the surrounding environment. Choose one application field — Architecture, Textiles/Fashion, or Robotics — and propose an application using cell-free systems that are functionally integrated into the material. Answer each of these key questions for your proposal pitch:

Write a one-sentence summary pitch sentence describing your concept. A paper-based, low-cost diagnostic tool that utilizes freeze-dried cell-free systems and RNA toehold switches to provide a visible color-change signal in the presence of Vibrio cholerae within 60 minutes.
How will the idea work, in more detail? Write 3-4 sentences or more. The device consists of a paper strip embedded with a freeze-dried cell-free transcription-translation (TX-TL) system and a synthetic “toehold switch” gene circuit. When a suspected water sample is dropped onto the paper, the water rehydrates the biological machinery, “waking it up.” If the specific RNA sequence of the Vibrio cholerae bacteria is present in the sample, it binds to the toehold switch, unfolding the RNA structure and allowing the ribosome to translate a reporter protein, such as LacZ or purple chromoprotein, which turns the paper from white to a distinct color.
What societal challenge or market need will this address? Cholera remains a major global health threat in resource-limited settings where traditional lab-based testing (like bacterial culture or PCR) is too slow, expensive, and dependent on a “cold chain” for refrigerated transport. This kit addresses the need for decentralized, rapid outbreak detection, allowing local health workers to confirm cases in the field and immediately initiate life-saving interventions like water chlorination and rehydration therapy.
How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

Activation: The limitation of needing water for activation is turned into a benefit here, as the liquid sample being tested serves as the rehydration agent for the freeze-dried reagents.

Stability: By incorporating lyoprotectants (such as trehalose) during the freeze-drying process, the enzymatic machinery is stabilized against high tropical temperatures, eliminating the need for refrigeration during shipping.

One-Time Use: To ensure the tool is economically viable despite being one-time use, the system is engineered onto cellulose paper, which is biodegradable and costs only cents per test, making it a scalable solution for mass surveillance during floods or humanitarian crises.