Week 9 Homework: Cell-Free Systems

Part 1: General Homework Questions

1. Advantages of Cell-Free Synthesis

Cell-free protein synthesis (CFPS) offers an “open” architecture, allowing for direct control over the chemical environment. Unlike in vivo methods, CFPS is not limited by cell toxicity or metabolic competition.

  • Toxic Protein Production: CFPS can produce proteins that would otherwise kill a living host cell.
  • High-Throughput Screening: It allows for the rapid testing of genetic libraries without the need for time-consuming cloning and cell culture.

2. Main Components

  • Cell Extract: The catalytic machinery (ribosomes, polymerases).
  • DNA Template: The genetic instructions.
  • NTPs & Amino Acids: The energy and building blocks.
  • Energy Regeneration: Essential for recycling ATP to sustain translation.

3. Energy Provision

Energy regeneration is critical because ATP is consumed rapidly and phosphate byproducts inhibit the reaction. The Creatine Phosphate/Creatine Kinase system is often used to maintain an ATP supply by transferring phosphate groups to ADP.

4. Prokaryotic vs. Eukaryotic Systems

  • Prokaryotic (E. coli): Best for high-yield, simple proteins like sfGFP.
  • Eukaryotic (Wheat Germ): Necessary for proteins requiring post-translational modifications, such as Human Insulin.

5. Membrane Protein Optimization

Membrane proteins often aggregate in aqueous environments. To optimize expression, I would use Nanodiscs or Liposomes in the reaction setup to provide a hydrophobic scaffold for co-translational insertion.

6. Troubleshooting Low Yield

  • Reason: Nuclease degradation. Fix: Use RNase inhibitors.
  • Reason: Magnesium imbalance. Fix: Perform a Mg(2+) titration.
  • Reason: Inefficient folding. Fix: Add molecular chaperones like GroEL/ES.

Part 2: Synthetic Minimal Cell Design (Kate Adamala)

Function: An environmental “Lead-Trapper” cell.

  • Input: Heavy metal ions (Lead/Pb).
  • Output: Lead sequestration inside the cell and a GFP signal.

Feasibility: Encapsulation is likely needed to physically isolate the lead from the environment in addition to cell-free.

  • Natural cell? Possible, but a minimal cell is safer as it cannot replicate or spread in the wild.

Design:

  • Membrane: POPC/Cholesterol phospholipids.
  • Encapsulation: E. coli PURE system for protein synthesis.
  • Communication: alpha-Hemolysin (αHL) pores (Gene: hlyA) to allow lead ions to enter the membrane.
  • Components: Gene 1: pbrR (Lead-responsive transcriptional regulator).
    • Gene 2: pcs (Phytochelatin synthase from S. pombe) to create lead-binding peptides.
  • Measurement: Fluorescence intensity from sfGFP tied to the PbrR promoter.

Part 3: Cell-Free Materials (Peter Nguyen)

Application Field: Textiles/Fashion

Pitch: A “living” athletic garment that detects dehydration in sweat and triggers a color-changing cooling response.

The Concept: The fabric is embedded with freeze-dried cell-free reactions containing synthetic gene circuits sensitive to sodium concentrations. When sweat salinity reaches a threshold, the circuit expresses a thermochromic pigment or activates an endothermic enzyme reaction. This addresses the risk of heatstroke in high-intensity athletes. To address stability, the reactions are encapsulated in trehalose-stabilized micro-beads that are only activated by the moisture in sweat.

Part 4: Mock Genes in Space Proposal (Ally Huang)

Background: Long-duration spaceflight leads to immune system dysregulation, specifically T-cell suppression. Understanding how immune-related genes, such as Interleukin-2 (IL-2), are transcribed in microgravity is vital for ensuring astronaut health on future Mars missions.

Molecular Target: Human Interleukin-2 (IL-2) promoter and gene sequence.

Target Relation: IL-2 is essential for T-cell proliferation. By using BioBits® to express IL-2 in space, we can determine if transcriptional machinery is hindered by microgravity or radiation independent of complex whole-cell physiology.

Goal: To determine if microgravity affects the yield of immune-related proteins.

  • Hypothesis: Transcriptional yield in space will be 20% lower than Earth controls due to altered molecular diffusion kinetics.
  • Reasoning: Microgravity alters fluid dynamics, potentially slowing the interaction between RNA polymerase and DNA templates.

Experimental Plan: Samples consist of freeze-dried BioBits® pellets containing IL-2 DNA. On the ISS, samples are rehydrated and placed in the P51 Molecular Fluorescence Viewer. A matching control experiment is run on Earth. Data is collected via fluorescence photography to measure protein production rates.

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