cell free system

General homework questions

  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.

The primary advantage of cell-free systems is the removal of the cell membrane, which eliminates the “black box” nature of cellular metabolism.

Flexibility and Control: Since the system is open, you can directly manipulate the reaction environment. You can add non-natural amino acids, adjust pH, or tune redox potential without worrying about maintaining cell viability. Unlike in vivo methods, you don’t need to worry about the metabolic burden on the host or the toxicity of the protein being produced.

Case 1: Toxic Proteins: Producing proteins that would normally kill a host cell (e.g., antimicrobial peptides or certain pore-forming toxins) is easy in CFPS because there is no “living” host to kill.

Case 2: Rapid Prototyping: CFPS allows for “pipette-and-test” cycles. You can test hundreds of genetic variants in hours without the time-consuming steps of transformation, cloning, and cell culture.

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

A standard cell-free reaction requires three main categories of components:

Component Role

  • Crude Extract (Lysate): Provides the “machinery”: ribosomes, aminoacyl-tRNA synthetases, translation factors, and often RNA polymerase.
  • Energy Solution: Contains an energy source (like phosphoenolpyruvate or glucose) and nucleotide triphosphates (ATP, GTP, UTP, CTP) to fuel the reaction.
  • Reaction Buffer: Contains essential ions ($Mg^{2+}$ and $K^{+}$), amino acids, and chemical additives to maintain pH and osmotic balance.
  1. 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.

Why it’s critical: Protein synthesis is energetically expensive. Every peptide bond requires the hydrolysis of multiple high-energy phosphate bonds. Without a regeneration system, the accumulated inorganic phosphate ($P_i$) inhibits the reaction, and ATP levels drop rapidly, leading to very low yields.

Method for Continuous Supply: One common method is the use of secondary energy substrates combined with specific enzymes. For example, using Creatine Phosphate and Creatine Kinase. The kinase transfers a phosphate group from creatine phosphate back to ADP, constantly replenishing the ATP pool.

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

The choice depends entirely on the complexity of the protein.

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

    • Protein Choice: GFP (Green Fluorescent Protein). It is a simple, robust protein that doesn’t require complex folding or glycosylation, making it ideal for the high-speed E. coli system.

  • Eukaryotic (e.g., CHO or Wheat Germ): Lower yields but capable of complex folding and PTMs (like glycosylation or disulfide bond formation).

    • Protein Choice: Human Erythropoietin (EPO). As a therapeutic glycoprotein, EPO requires specific glycosylation patterns to be biologically active, which only a eukaryotic system (like CHO cell extract) can provide.
  1. 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. In a standard aqueous cell-free mix, they often misfold or aggregate because there is no lipid environment to stabilize their transmembrane domains.

Design Strategy:

  1. Supplement with Surfactants/Lipids: Add nanodiscs, liposomes, or mild detergents directly to the reaction. This provides a “home” for the protein as it is translated.

  2. Addressing the Challenge: To ensure proper orientation, use pre-formed liposomes. As the ribosome translates the mRNA, the hydrophobic regions of the protein can spontaneously insert into the lipid bilayer of the liposome.

  3. 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.

Reason for Low Yield Troubleshooting Strategy

  • mRNA Degradation: Add RNase inhibitors or ensure all equipment is RNase-free. You can also check mRNA stability by running a gel.
  • Magnesium Imbalance: Perform a Magnesium Titration. CFPS is extremely sensitive to Mg{2+} concentration; test a range (e.g., 8mM to 20mM) to find the “sweet spot” for your specific template.
  • Codon Bias: If using a human gene in an E. coli extract, the tRNA pools may not match. Use codon-optimized DNA or supplement the reaction with specialized tRNA mixtures (e.g., Rosetta-style extracts).

Homework question from Kate Adamala:

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

  1. Pick a function and describe it.

a. What would my synthetic cell do? What is the input and what is the output?

My synthetic cell will function as a Smart Antioxidant Biosensor. It is designed to detect environmental oxidative stress and respond by producing and releasing protective enzymes.

  • Input: High concentrations of Reactive Oxygen Species (ROS), specifically Hydrogen Peroxide ($H_2O_2$).
  • Output: The production and secretion of the enzyme Catalase.

b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

While the chemical reactions for transcription and translation (Tx/Tl) would occur in an open solution, the system would lack stability. Without encapsulation in a lipid bilayer, the DNA, enzymes, and ribosomes would be immediately diluted and degraded in a complex environment (like a biological tissue). Encapsulation is essential to maintain a “protected reaction space.”

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

Yes, it could. However, a synthetic cell is a “minimal” and safer alternative. Natural cells have complex metabolic backgrounds that might interfere with the specific task, and they pose a risk of uncontrolled replication. A synthetic cell is a programmable, non-living machine that performs only the desired function.

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

The goal is for these cells to act as localized therapeutic agents. When they encounter an area of inflammation (marked by high ROS), they “turn on” their internal factory, produce Catalase, and neutralize the H2O2, thereby preventing oxidative damage to surrounding healthy cells.

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

a. What would be the membrane made of?

The membrane will be a Lipid Bilayer composed of POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) for basic structure and Cholesterol to enhance membrane stability and mimic human cell fluidity.

b. What would you encapsulate inside?

  • Transcriptional/Translational Machinery: Ribosomes, T7 RNA Polymerase, amino acids, and tRNAs.

  • Energy Regeneration System: Creatine Phosphate and Creatine Kinase to ensure a steady supply of ATP.

  • Genetic Material: A custom-designed plasmid DNA.

c. Which organism will your Tx/Tl system come from?

I will use a Bacterial (E. coli) system.

Reasoning: Bacterial extracts (like PURE system or S30 lysate) are much more robust and offer significantly higher protein yields compared to mammalian systems. Speed is critical when the goal is to neutralize oxidative stress quickly.

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

Communication will occur via Membrane Channels. Since the Catalase enzyme is too large to diffuse through the lipid bilayer, I will incorporate a pore-forming protein into the membrane to allow the output to exit the synthetic cell.

  1. Experimental details

a. List all lipids and genes.

  • Lipids: POPC and Cholesterol.
  • Gene 1 (The Sensor): The oxyS promoter. This is an E. coli promoter that is specifically activated in the presence of oxidative stress.
  • Gene 2 (The Effector): The katG gene, which encodes for the enzyme Catalase to break down H2O2.
  • Gene 3 (The Channel): The alpha-Hemolysin (hlyA) gene. This produces a small pore (1.4 nm) in the membrane, allowing the produced enzymes to be released into the environment.

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

I will use a two-step verification process:

  1. Fluorescence Reporting: I will include a GFP (Green Fluorescent Protein) gene under the same oxyS promoter. If the synthetic cells glow green when H2O2 is added, it proves the sensing and translation mechanisms are working.
  2. Activity Assay: I will use an Amplex Red Assay. This reagent changes color in the presence of H2O2. If the color intensity decreases after adding my synthetic cells to the sample, it confirms that Catalase is being successfully produced and released to neutralize the oxidant.

Homework question from Peter Nguyen:

I chose Textiles/Fashion because it allows us to turn everyday clothing into a life-saving diagnostic tool.

  • Pitch Sentence: I propose the “Bio-Active Rescue Suit,” a garment embedded with freeze-dried cell-free sensors designed to detect toxic environmental chemicals and change color to warn the wearer of invisible dangers.

  • How it works: The cell-free reaction components (ribosomes, DNA templates, and energy buffer) are freeze-dried onto the textile fibers. When the fabric is exposed to a specific liquid or moisture containing a target contaminant (like heavy metals or nerve agents), the water rehydrates the system. This triggers the transcription and translation of a “reporter protein” like mCherry or GFP, causing a visible color change on the clothing surface within minutes.

  • Societal Challenge: This addresses the need for real-time, wearable safety monitoring for industrial workers, first responders, and soldiers who often lack bulky diagnostic equipment in high-risk zones.

  • Addressing Limitations: To handle one-time use and stability, the sensors are designed as modular, replaceable “bio-patches” that can be attached to the suit. To prevent accidental activation by rain, the patches are covered by a semi-permeable membrane that only allows specific target molecules to pass through while keeping pure water out.

Homework question from Ally Huang:

This proposal uses the BioBits® system to solve a critical healthcare challenge for long-term space travel.

  1. A major challenge for deep-space missions is “Radiation-Induced Oxidative Stress.” High-energy cosmic rays damage human DNA, leading to a build-up of reactive oxygen species (ROS) that can cause cancer or organ failure. Currently, we cannot monitor an astronaut’s cellular response to radiation in real-time. Developing a portable, low-resource method to detect cellular stress markers is vital for protecting astronaut health during a journey to Mars. This research is significant because it paves the way for “on-demand” diagnostics in environments where traditional labs are impossible.
  2. The genetic target is the human HMOX1 (Heme Oxygenase-1) promoter fused to a green fluorescent protein (GFP) reporter gene. HMOX1 is a primary biomarker for oxidative stress.
  3. In space, radiation causes systemic inflammation. The HMOX1 gene is naturally up-regulated by the body to fight this stress. By using the HMOX1 promoter in a BioBits® system, we can create a “living” sensor that mimics the human body’s response. If an astronaut’s blood or saliva sample contains stress-induced signaling molecules, the BioBits® system will activate the promoter, indicating that the radiation levels have reached a dangerous threshold for human biology.
    • Hypothesis / Research GoalHypothesis: I hypothesize that a freeze-dried BioBits® cell-free system, utilizing a stress-responsive promoter, can accurately detect biomolecular markers of radiation damage in a microgravity environment as effectively as it does on Earth.
    • Reasoning: Microgravity affects molecular diffusion and protein folding. By testing this system on the ISS, we can determine if cell-free sensors remain reliable diagnostic tools for astronauts. My goal is to validate that the “Bio-Bits + P51 Viewer” setup can provide a “Yes/No” visual signal for cellular damage without needing complex liquid handling.
  5. I will test freeze-dried BioBits® pellets containing the HMOX1-GFP reporter DNA.
    • Samples: One set of pellets will be activated with a “stressed” sample (containing H2O2 or radiation-mimetic chemicals), and the Control set will be activated with pure water.
    • Measurements: After incubation, I will use the P51 Molecular Fluorescence Viewer to measure the intensity of green light produced.
    • Data: I will record the time it takes for a visible signal to appear and compare the brightness of the ISS samples against Earth-based controls to account for microgravity effects.