Week 9: Cell-Free Systems

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 and Control: Since there is no cell membrane, the system is an open environment, where we can directly manipulate concentrations of substrates, add non-canonical amino acids, or introduce specific inhibitors/activators with no worries about cellular transport or toxicity.
• Case 1: Toxic Proteins: Many proteins, such as antimicrobial peptides or certain enzymes, are lethal to host cells. CFPS allows the production of these proteins.
• Case 2: Rapid Prototyping: In vivo methods need time-consuming cloning, transformation, and cell culture. CFPS use’s linear DNA as a template, reducing the time from days to hours.

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

• Cell Extract (Crude Lysate): It contains ribosomes, aminoacyl-tRNA synthetases, translation factors, and tRNAs.
• Energy and Buffer Systems: This includes ATP and GTP , an energy regeneration substrate (like Phosphoenolpyruvate), and essential ions (Mg2+ and K+) to maintain enzymatic activity and pH.
• Genetic Template and Building Blocks: DNA provides the instructions, while the 20 standard amino acids provide the raw material for the protein chain.

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.

Protein synthesis is energetically expensive. ATP is consumed rapidly for amino acid activation and ribosome movement, and it is also naturally degraded by other enzymes in the extract.

• Why is it critical: If ATP is depleted and ADP/inorganic phosphate builds up, the reaction stalls, leading to very low yields.
• Method for Continuous Supply: By adding a high-energy phosphate donor like creatine phosphate and the enzyme creatine kinase, we can have the system continuously re-phosphorylate ADP back into ATP during the reaction.

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

The choice of system depends on the complexity of the target protein.

• Prokaryotic (e.g., E. coli): These are high-yield, cost-effective, and fast.
  • Protein Choice: GFP (Green Fluorescent Protein). Since GFP does not require complex post-translational modifications (PTMs) to function, the E. coli system is ideal for producing it in large quantities quickly.
• Eukaryotic (e.g., Wheat Germ, CHO cells): These are slower, more expensive but allow for proper folding of complex proteins and PTMs.
  • Protein Choice: Human Tissue Plasminogen Activator (tPA). This protein requires multiple disulfide bonds and glycosylation to be active, which eukaryotic machineries can provide.

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.

Membrane proteins are very difficult because they have hydrophobic regions that aggregate in the watery environment of test tubes.

• Design/Setup: Introduce Liposomes directly into the CFPS reaction.
• Challenges: The primary challenge is misfolding and precipitation.
• Addressing it: By providing a lipid bilayer during the translation process (co-translational insertion), the protein can embed its hydrophobic domains into the lipids as it is being synthesized, mimicking its natural environment.

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.

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 your synthetic cell do? What is the input and what is the output? b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation? c. Could this function be realized by genetically modified natural cell? d. Describe the desired outcome of your synthetic cell operation.

2. Design all components that would need to be part of your synthetic cell. a. What would be the membrane made of? b. What would you encapsulate inside? Enzymes, small molecules. c. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian) d. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

3. Experimental details: a. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.) b. How will you measure the function of your system?

Refer to the example solution given in the website if needed.

1. Function and Logic:

• What would my synthetic cell do?
  • It will act as a localized logic gate. It senses an acidic, high-lactate environment (Input) and, in response, synthesizes and releases a pore-forming toxin (Output) to induce lysis in adjacent tumor cells.
• Input:
  • High extracellular L-Lactate and low pH.
• Output:
  • Melittin (a potent cytolytic peptide) and Cy5 Dye (for visual tracking/imaging).
• Could this be realized by cell-free Tx/Tl alone?
  • No. Encapsulation is vital to prevent Melittin from diffusing away and damaging healthy tissue. The vesicle will ensure the toxin is only released at the specific site of the trigger.
• Could this be realized by a genetically modified natural cell?
  • While possible, natural cells often face “exhaustion” or can be suppressed by the tumor’s immune-evasive signals. An Synthetic Minimal Cell doesn’t have receptors for immune suppression, making it more robust in the harsh tumor microenvironment.
• Desired Outcome:
  • Selective destruction of tumor cells with minimal off-target effects on healthy cells.

2. Component Design:

• Membrane:
  • DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DSPE-mPEG. The PEGylation (mPEG) allows the SMC to circulate in the bloodstream longer without being cleared by the immune system.
• Encapsulation:
  • Mammalian or Bacterial Tx/Tl machinery.
    • LldR (Lactate-responsive regulator).
    • Genetic template for the toxin.
• Organism Source:
  • Bacterial (E. coli) is suitable for the high-yield production of small peptides, like Melittin. But, using a PURE system (Protein synthesis Using Recombinant Elements) is even better here as it contains only the essential translation factors, reducing the chance of metabolic interference.
• Communication:
  • The membrane will contain a gated protein channel. We can express a channel that allows the toxin to exit only after the sensor is triggered.

3. Experimental Details:

4. Measuring Function:

• Lactate Response Assay:
  • In a lab setting, place the SMCs in media with varying concentrations of L-lactate (0mM to 20mM).
  • Use HPLC or Mass Spectrometry to measure how much Melittin is released into the supernatant.
• Cytotoxicity Co-culture:
  • Place the SMCs in a dish with Ovarian Cancer cells (SKOV3).
  • Use a Live/Dead stain (like Calcein AM/Ethidium Homodimer) and observe under a confocal microscope.
  • Cancer cell death should be seen only in the presence of lactate-triggered SMCs.
• Vesicle Tracking:
  • Use In Vivo Imaging Systems (IVIS) to track the Cy5 fluorescence in a mouse model.

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

A “bio-reactive” building skin composed of freeze-dried, cell-free synthetic cells integrated into porous ceramic tiles that detect airborne toxins and respond by secreting neutralizing enzymes to purify the local air.

The ceramic facade tiles are manufactured with a network of microfluidic channels having paper-based, freeze-dried cell-free (CF) extracts. These extracts are programmed with a genetic AND gate: they require the presence of a pollutant (such as Formaldehyde or Volatile Organic Compounds (VOCs)) and atmospheric moisture to activate. When the pollutant diffuses into the tile, it binds to a specific protein (like the frmR regulator for formaldehyde) encapsulated within the synthetic cells. This triggers the TX-TL machinery to synthesize a degradative enzyme (such as Formaldehyde Dehydrogenase), which is then secreted onto the surface of the tile to chemically break down the toxin into harmless byproducts like formate and water.

This addresses the growing crisis of urban air pollution. In dense cities, indoor and immediate outdoor air quality is often compromised by industrial emissions and off-gassing from construction materials. Current solutions (like HEPA filters or HVAC systems) are expensive, energy-intensive and require mechanical maintenance. This system provides a passive, carbon-neutral way to scrub the air, essentially turning the building itself into a biological lung that protects the health of its occupants and the surrounding community.

• Activation & Moisture: We can utilize a hydrophobic wax coating that melts or becomes permeable only at specific temperatures or humidity levels, or use a “peel-and-activate” surface where the cell-free system remains dormant until the first rainfall or a controlled misting event.
• Stability: Freeze-drying (lyophilization) allows the TX-TL components to remain stable at room temperature for over a year. By incorporating trehalose (a sugar that protects molecular structures), we can ensure the ribosomes and enzymes remain functional while dormant.
• Sustainability & Re-use: To move past one-time use, the facade tiles can be designed with replaceable cartridges. Similar to an ink cartridge in a printer, the “bio-active” paper strips can be swapped out once the enzymatic capacity is exhausted, or the tiles can be “re-seeded” with a fresh aerosolized cell-free mix.

Homework question from Ally Huang:

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in my talk, the Genes in Space competition challenges students to consider how biotechnology, including cell-free reactions, can be used to solve biological problems encountered in space. While the competition is limited to only high school students, your assignment will be to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space!

For this particular assignment, your proposal is required to incorporate the BioBits® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/ .

a. Provide background information that describes the space biology question or challenge you propose to address. Explain why this topic is significant for humanity, relevant for space exploration, and scientifically interesting. (Maximum 100 words) b. Name the molecular or genetic target that you propose to study. Examples of molecular targets include individual genes and proteins, DNA and RNA sequences, or broader -omics approaches. (Maximum 30 words) c. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) d. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) e. Outline your experimental plan - identify the sample(s) you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, etc. (Maximum 100 words)

Extended stays in microgravity cause astronauts to lose bone mass at a rate of 1–2% per month, a condition known as spaceflight-induced osteopenia. This is a critical barrier, as it increases fracture risk and renal stones. While exercise helps, it doesn’t fully stop the loss. This challenge is significant because it mimics Earth-based osteoporosis, offering a unique “accelerated” model for bone research. Studying this in space allows us to test rapid-response biotechnologies that could eventually provide on-demand therapeutic proteins to maintain astronaut health during years of isolation from Earth.

• Molecular/Genetic Target:

  • The target is the Sclerostin (SOST) gene and its protein product. Sclerostin is a key inhibitor of bone formation; when levels are high, bone building stops.

• Relation to Space Biology:

  • Microgravity triggers an upregulation of the SOST gene in osteocytes, leading to an overproduction of Sclerostin. This stop signal prevents osteoblasts from creating new bone tissue, resulting in a net loss of density. By focusing on this target, we can monitor this switch that causes bone loss. Using a cell-free system to produce and study Sclerostin-binding aptamers or inhibitory proteins allows us to investigate ways to block this signal directly in the space environment, moving away from simple monitoring toward active biological countermeasures.

• Hypothesis:

  • A freeze-dried BioBits® system can be used to synthesize a functional Sclerostin-sensing fluorescent biosensor in microgravity, providing a real-time tool to measure the effectiveness of bone-loss inhibitors.

• Reasoning:

  • Currently, analyzing protein levels requires sending samples back to Earth. I propose creating a synthetic genetic circuit where a Sclerostin-binding aptamer is linked to the production of a fluorescent protein (like mCherry) in a BioBits® reaction. If the Sclerostin levels are high, the fluorescence will be quenched or activated depending on the circuit design. This provides an on-the-spot diagnostic. Because BioBits® is freeze-dried, it is lightweight and shelf-stable, making it ideal for this experiment in the resource-limited environment of the space stations.

• Experimental Plan:

  • We will use BioBits® pellets containing the genetic circuit for the Sclerostin biosensor.
    • Test Samples: Synthetic Sclerostin protein added to the BioBits® reaction to simulate astronaut serum levels.
    • Controls: A positive control (BioBits® + constitutive GFP DNA) and a negative control (BioBits® + water).
    • Measurement: Samples will be incubated in the miniPCR® and visualized using the P51 Fluorescence Viewer.
    • Data: We will record the intensity of fluorescence, which correlates to Sclerostin concentration, proving cell-free systems can perform complex diagnostic sensing in orbit.

Thank You!