Week 9 HW: Cell Free System

Homework Part A: General and Lecturer-Specific 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.

Advantages:1.Flexibility and Control: You can directly adjust the reaction system, such as changing pH, ion concentration, or adding non-natural amino acids, without worrying about “killing” cells.2.Openness: The system is open; you can monitor the reaction process in real time or directly add linear DNA without constructing complex plasmids.

Applications:Synthesis of Toxic Proteins: Some proteins kill host cells but can be safely produced in a cell-free system.

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

Crude Extract: Contains ribosomes, RNA polymerase, and translation factors (the “factories” for protein synthesis).

Energy Source: Such as phosphoenolpyruvate (PEP), providing the driving force for the reaction.

Amino Acids: The raw materials for protein synthesis.

Cofactors and Salts: Such as Mg²⁺ and K⁺, maintaining enzyme activity.

DNA Template: Contains instructions that encode the target protein.

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.

Importance: Protein synthesis is an extremely energy-intensive process. If ATP is depleted, the reaction will immediately cease, and the accumulation of byproducts (such as inorganic phosphate) will inhibit the reaction.

Methods to ensure a continuous supply of ATP: Use an energy regeneration system: Add creatine phosphate and creatine kinase. Creatine phosphate continuously converts ADP back into ATP, maintaining stable energy levels.

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

1.Structure

The prokaryotic system has a very simple structure; transcription and translation occur almost simultaneously, resulting in an extremely fast reaction rate. The eukaryotic system has a more complex mechanism, better mimicking the internal environment of complex organisms.

2.Protein folding and modification capabilities

Prokaryotic systems typically lack the ability to handle complex folding. Eukaryotic systems, on the other hand, are equipped with various “helpers” (molecular chaperones) that enable them to perform crucial post-translational modifications (such as glycosylation and disulfide bond formation), which are essential for proteins to exhibit biological activity.

5.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.

Cause: Template DNA degradation. Strategy: Check DNA purity, or add RNase inhibitors to prevent mRNA degradation.

Cause: Energy depletion or pH shift. Strategy: Optimize the energy regeneration system, or use dialysis to continuously replenish substrate and remove metabolic waste.

Cause: Protein misfolding leading to degradation. Strategy: Lower the reaction temperature (e.g., from 37°C to 25°C), or add molecular chaperones to assist folding.

Homework question from Kate Adamala

Synthetic Minimal Cell Design: The Hotpot Odor Eliminator（Gemini Help）

1. Function & Operation Description

What it would do: This synthetic cell is designed as a fabric spray to eliminate the stubborn “hotpot smell” from clothes. The signature smell of hotpot comes primarily from volatile organic compounds, notably sulfur-containing compounds (like dimethyl sulfide from garlic and onions) and pyrazines (from roasted spices and oils). This synthetic cell senses these oily odor molecules on the fabric, actively absorbs them, and enzymatically neutralizes them into completely odorless molecules.

Input: Dimethyl Sulfide (DMS) and Pyrazines (The dominant volatile organic compounds causing the hotpot odor).

Output: Odorless sulfoxides and cleaved non-volatile fragments (completely neutral scents).

2. Structural & Component Design Membrane Composition The membrane needs to interact with clothing fibers and oily food odors: POPC (a standard phospholipid) mixed with DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). Reasoning: DOPE increases the hydrophobicity and fluidity of the membrane, allowing it to easily trap and dissolve the greasy, airborne hotpot oil droplets present on the fabric.

Encapsulation Inventory Machinery: A robust Bacterial Cell-Free Expression System (like E. coli PURExpress). Why bacterial instead of mammalian this time? Because we are working in an open-air environment on clothes, we need a system that is highly stable, cheap to produce, and does not require complex mammalian temperature/co-factor controls. Genetic Blueprint: Plasmids encoding the odor-degrading enzymes. Internal Fuel: A pre-packaged mix of 20 amino acids, ATP/GTP energy molecules, and magnesium salts.

3. Experimental Details List of Lipids POPC (Basic membrane matrix) DOPE (Hydrophobic lipid to attract hotpot oil vapors) Specific Genes (The Deodorizing Circuit) We will encapsulate plasmids encoding two primary enzymes under a strong, constitutive T7 promoter: snoA (Sulfur Oxygenase/Reductase gene): Derived from sulfur-oxidizing bacteria. This enzyme converts the pungent Dimethyl Sulfide (DMS) into odorless dimethyl sulfoxide (DMSO). pyrD (Pyrazine-degrading Monooxygenase gene): This enzyme targets the roasted, nutty pyrazines from hotpot spices, breaking their aromatic ring structures apart and rendering them completely scentless.

4. Functional Measurement & Readout To prove the “Hotpot Protocell Spray” actually works in the lab: Gas Chromatography-Mass Spectrometry (GC-MS) Headspace Analysis: We expose a piece of fabric to hotpot steam, spray it with our synthetic cells, and seal it in a glass vial. We then use GC-MS to measure the concentration of volatile organic compounds in the air above the fabric over time. A sharp decrease in the peak intensities of dimethyl sulfide and pyrazines proves the system is destroying the odor. Colorimetric Enzyme Assay: We can engineer the snoA or pyrD reaction to trigger a secondary dye change inside the liposome (for example, using a pH-sensitive indicator dye that changes color when the acidic breakdown products accumulate). If the spray slightly shifts color (e.g., from clear to faint blue) upon contact with hotpot odor, it provides a visual confirmation that the人造细胞 successfully “ate” the smell.

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. How will the idea work, in more detail? Write 3-4 sentences or more. What societal challenge or market need will this address? How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

Summary Pitch Develop a “smart protective suit” with built-in cell-free sensors that can detect toxic chemicals or pathogens in the air in real time through color changes, thus protecting the wearer’s safety.

Detailed Mechanism This concept encapsulates a freeze-dried, cell-free reaction system (containing DNA sensors, ribosomes, and energy substances) within microcapsules of fibers. When the wearer is exposed to specific contaminants (such as heavy metals or specific bacterial signaling molecules), these molecules penetrate the microcapsules. These molecules act as “switches,” initiating the transcription and translation of DNA templates to synthesize chromogenic proteins (such as red fluorescent protein). Ultimately, a visible color change occurs on the surface of the garment, thus triggering an alarm.

Societal Challenge This addresses the issue of hidden threats to occupational safety. Chemical workers, frontline epidemic prevention personnel, and miners are frequently exposed to colorless and odorless hazardous gases or pathogens. Existing electronic sensors are often bulky and require batteries, while this biosensor integrated into fabric requires no power source, is lightweight, and provides full-body monitoring.

Homework question from Ally Huang

Rapid detection of radiation damage in space environment

Background Information During long-term deep-space exploration, astronauts are exposed to intense cosmic radiation, which may cause DNA damage and increase the risk of cancer. In a resource-constrained space environment, real-time and convenient monitoring of the biological effects induced by radiation is crucial for the health of astronauts.

Scientific Interest Traditional live-cell detection relies on complex culture equipment. By using the freeze-dried cell-free system (BioBits®), we can convert the molecular changes induced by radiation into visible fluorescence signals without maintaining the survival of live cells, providing immediate warnings for space biological safety.

Molecular or Genetic Target Protein expression driven by the p53 response element. P53 is the crucial “genomic guardian” in the human body, and its activity significantly increases in response to DNA damage.

Hypothesis / Research Goal Objective: To verify whether the BioBits® system can function as a reliable, non-temperature-controlled biosensor in a microgravity environment, by detecting the level of p53 protein to indicate the degree of DNA damage.

Hypothesis: We hypothesize that the fluorescence intensity triggered by the p53 protein is positively correlated with the radiation dose.

Reasoning: In the Earth laboratory, the p53-mediated reaction is a standard biomarker for cellular stress. The freeze-dried BioBits® reaction components have extremely high stability and are suitable for storage on the International Space Station (ISS). By combining with the P51 fluorescence imaging microscope, astronauts can quickly determine the radiation exposure risk by observing the color brightness without having to send the samples back to Earth for analysis, significantly reducing the response time.