Week 9 — Cell-Free Systems

Part A: General and Lecture- Specific

1. 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.
  • Flexibility & Control: Unlike living cells, CFPS is an “open” system. You can directly manipulate the reaction environment—adjusting pH, redox potential, or adding non-natural amino acids—without worrying about maintaining cell viability.
  • Speed: It bypasses the time-consuming steps of transformation, cell culture, and scale-up, allowing for results in hours rather than days.
  • Beneficial Cases:
    • Toxic Proteins: Expressing proteins that would kill a living host cell (e.g., certain antimicrobial peptides).
    • Rapid Prototyping: Testing many different genetic designs quickly in a high-throughput format.
2. Describe the main components of a cell-free expression system and explain the role of each component.
  • Cell Extract: Provides the molecular “machinery,” including ribosomes, tRNAs, and initiation/elongation factors.
  • Energy Source: Molecules like phosphoenolpyruvate (PEP) or creatine phosphate used to regenerate ATP and GTP.
  • Amino Acids: The essential building blocks for synthesizing the protein chain.
  • DNA Template: The genetic instructions (plasmid or linear PCR product) for the target protein.
  • Buffer/Salts: Maintains optimal pH and ionic strength (especially Mg2+ and K+) required for ribosomal function.
3. 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.
  • Criticality: Energy is consumed rapidly during transcription and translation. Without a regeneration system, the reaction stops once the initial ATP pool is depleted, leading to very low yields.
  • Method: Use an enzymatic substrate system, such as the Creatine Phosphate/Creatine Kinase system, which transfers a phosphate group back to ADP to regenerate ATP in situ.
4. 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 complex post-translational modifications (PTMs).
    • Protein: GFP (Green Fluorescent Protein) for simple reporting or biosensing where complex folding isn’t required.
  • Eukaryotic (e.g., CHO or Wheat Germ): Lower yield but capable of complex folding and PTMs like glycosylation.
    • Protein: Human Insulin, which requires specific disulfide bond formation and folding pathways not present in bacteria.
5. 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.
  • Challenges: Membrane proteins are hydrophobic and often aggregate or misfold when synthesized in an aqueous cell-free mix without a lipid environment.
  • Optimization Strategy: Integrate synthetic lipid bilayers
6. 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: - Reason: Presence of RNases or DNases in the extract.
    • Strategy: Use high-purity DNA and supplement the reaction with RNase inhibitors.
  • Energy Depletion: - Reason: Fast consumption of ATP/GTP.
    • Strategy: Increase the concentration of the energy buffer or use a dialysis-based continuous-exchange system.
  • Codon Bias: - Reason: The DNA sequence uses codons that are rare in the organism the extract was made from.
    • Strategy: Use a codon-optimized gene sequence or supplement the reaction with a mixture of rare tRNAs.

Homework question from Kate Adamala

1. Design an example of a useful synthetic minimal cell as follows:

  1. Pick a function and describe it.
    • Function: DermLogic is a dual-channel AND-gate biosensor designed for point-of-care HPV detection and therapeutic antisense RNA delivery.
    • Input/Output: The input is extracellular HPV L1 and E6/E7 RNA sequences; the output is a dual-fluorescence signal (GFP/mCherry) and the synthesis of therapeutic antisense RNA.
  2. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
    • Yes, the core logic functions in a bulk cell-free mix. However, encapsulation is required for the “therapeutic patch” vision to protect the RNA payload from degradation and to concentrate the reagents for faster kinetics.
  3. Could this function be realized by genetically modified natural cell?
    • It is difficult. Natural cells have complex innate immune responses (like interferon pathways) that might interfere with or degrade synthetic RNA logic circuits and antisense outputs.
  4. Describe the desired outcome of your synthetic cell operation.
    • A low-cost, decentralized tool that stratifies HPV risk and simultaneously produces a customized therapeutic response without a cold chain.

2. Design all components that would need to be part of your synthetic cell.

  1. What would be the membrane made of?
    • A robust, shelf-stable lipid bilayer composed of POPC and Cholesterol, or potentially a polymeric/hybrid vesicle for better stability during lyophilization.
  2. What would you encapsulate inside?
    • BL21 DE3 Lysate, the pDermLogic-v1 plasmid, T7 RNA Polymerase, amino acids, and an energy regeneration system (PEP/ATP).
  3. Which organism your Tx/Tl system will come from?
    • Bacterial (E. coli) is preferred. It is highly efficient for T7-driven transcription and the toehold switches are optimized for bacterial ribosomes.
  4. How will your synthetic cell communicate with the environment?
    • Through expressed membrane pores like alpha-hemolysin (aHL). These allow the viral RNA “triggers” to enter the cell and the fluorescent/therapeutic outputs to be detected or released.

3. Experimental details

  1. List all lipids and genes.
    • Lipids: POPC, Cholesterol.
    • Genes: sfGFP (Channel A reporter), mCherry (Channel B reporter), and a custom antisense RNA sequence targeting the HPV E6/E7 junction.
  2. How will you measure the function of your system?
    • Using a dual-channel fluorescence readout (Spark Plate Reader) to monitor the AND-gate logic, and denaturing PAGE gel electrophoresis to verify the production of the ~21 nt antisense RNA.

Homework question from Peter Nguyen

  • Pitch: “Bio-Sensing Textiles: A garment that changes color upon detecting hazardous environmental pathogens.”
  • How it works: Freeze-dried cell-free systems containing a specific RNA-based biosensor (riboswitch) are embedded into fabric fibers. Upon exposure to a specific pathogen and moisture (sweat or atmospheric water), the system rehydrates, triggers the sensor, and expresses a chromoprotein that visibly stains the fabric.
  • Societal Challenge: This addresses the need for passive, wearable safety monitoring for healthcare workers or soldiers in environments with invisible biological threats.
  • Addressing Limitations: The “one-time use” nature is addressed by making the sensor a disposable patch integrated into the garment; activation is solved by leveraging inherent moisture or the user’s perspiration as the rehydration trigger.

Homework question from Ally Huang

  1. Background Information: Microgravity and cosmic radiation cause significant muscle atrophy and DNA damage in astronauts. Monitoring real-time protein expression in space is difficult due to bulky equipment. BioBits® allows for rapid diagnostic tests on the ISS with a minimal footprint.
  2. Target: Myostatin (MSTN) protein levels, which are key regulators of muscle growth and indicators of muscle wasting.
  3. Relation to Space Biology: Tracking Myostatin levels allows researchers to quantify the rate of muscle degradation in microgravity. By using a cell-free biosensor, we can monitor these levels in real-time without needing to return samples to Earth.
  4. Hypothesis/Research Goal: Hypothesis: BioBits® can be engineered to produce a fluorescent signal proportional to the concentration of Myostatin mRNA. The goal is to create a “just-add-sample” diagnostic kit for astronauts to monitor their physical health during long-duration missions.
  5. Experimental Plan: We will test astronaut saliva samples. Control: Rehydrated BioBits® with a known concentration of MSTN DNA. Experimental: BioBits® rehydrated with the astronaut’s sample. Data will be collected using the P51 Molecular Fluorescence Viewer to observe the intensity of the green fluorescence.

Part B: Final Project Integration — DermLogic

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Aim 1: Cell-Free Logic Validation

The core of my final project, DermLogic, relies on the E. coli BL21 DE3 cell-free system to execute a complex molecular AND-gate. This week’s focus on cell-free systems directly informs my strategy for:

  • Signal Processing: Using dual-channel toehold switches (L1-GFP and E6/E7-mCherry) to stratify HPV risk.
  • On-Demand Therapeutics: Leveraging the “open” nature of cell-free systems to synthesize ~21 nt antisense RNA molecules immediately upon pathogen detection.

Lyophilization Strategy

Following the principles of BioBits®, DermLogic is designed to be a shelf-stable, “just-add-water” (or sample) diagnostic. My validation plan for Aim 1 includes:

  1. Cryoprotectant Optimization: Testing a mix of 100mM Trehalose and 0.1% BSA to ensure the T7 RNA Polymerase and ribosomes remain functional after freeze-drying.
  2. Stability Testing: Comparing the fluorescence kinetics of fresh vs. rehydrated pellets using the Spark Plate Reader to calculate signal retention.

Key Component: By encapsulating this reaction in a POPC/Cholesterol lipid bilayer with alpha-hemolysin pores, the system transforms from a bulk reaction into a “synthetic cell” capable of localized therapeutic delivery.