Week 9 HW: Cell-Free Systems

General and Lecturer-Specific Questions

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.

• Cell-free protein synthesis has a big advantage over in vivo methods because it gives you direct control over the reaction environment without needing to keep cells alive. You can precisely tune things like DNA concentration, energy sources, cofactors, salts, and even add or remove specific components in real time, which is much harder to do inside living cells where metabolism and regulation get in the way. It’s also faster since you skip cloning, transformation, and cell growth steps. This makes it especially useful for expressing toxic proteins that would kill or stress cells, and for rapid prototyping or screening large libraries of genetic constructs where you want quick, iterative testing without waiting on cultures to grow.

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

• A cell-free expression system is mainly made up of a cell extract, a DNA template, and a reaction mix that supports transcription and translation. The cell extract provides the core molecular machinery, like ribosomes, tRNAs, aminoacyl-tRNA synthetases, transcription and translation factors, which are all needed to actually make protein. The DNA template contains the gene of interest along with the regulatory sequences needed for expression. The reaction mix supplies the raw materials and energy needed to drive the system, including amino acids, nucleotides, salts, cofactors, ATP regeneration components, and buffering agents to keep conditions stable. Together, these components recreate the basic protein production machinery of a cell, but in a much more controllable format.

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.

• Energy provision and regeneration are critical in cell-free systems because transcription and translation burn through ATP and GTP fast, so without a way to replenish that energy, protein synthesis stalls. Since there are no living cells to continuously regenerate energy through metabolism, the reaction depends entirely on whatever energy system you build into it. Basically, if the reaction runs out of usable energy, the whole system stalls, so energy regeneration is what keeps protein production going for longer and improves overall yield. One common way to maintain ATP supply is to include an energy regeneration substrate such as phosphoenolpyruvate (PEP), which can be used to help regenerate ATP during the reaction. In the reaction, PEP transfers a phosphate group to ADP through the enzyme pyruvate kinase, which regenerates ATP that can then be used to keep transcription and translation going.

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

• Prokaryotic and eukaryotic cell-free systems each have different strengths depending on the type of protein being produced. Prokaryotic systems, like E. coli extracts, are usually faster, cheaper, and great for making simple proteins that do not need complex folding or post-translational modifications. In contrast, eukaryotic cell-free systems are better for proteins that require more advanced folding, disulfide bond formation, or modifications that bacteria cannot do well. For a prokaryotic system, a strong candidate would be Luz (luciferase) from the fungal bioluminescence pathway, since it is a relatively compact enzyme that folds well in bacterial extracts and does not require eukaryotic post-translational modifications; producing it cell-free would allow rapid screening of variants and direct assay of luminescence activity by simply adding the 3-hydroxyhispidin substrate to the reaction. For a eukaryotic system, a suitable target would be H3H (hispidin-3-hydroxylase) or another upstream enzyme in the caffeic acid–to–luciferin pathway, since these fungal oxidative enzymes often depend on proper folding, cofactor incorporation, and a eukaryotic redox environment to remain active. Expressing the pathway enzymes in their appropriate systems enables modular prototyping of the bioluminescence circuit before committing to stable plant transformation.

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.

• To optimize expression of a membrane protein in a cell-free system, I would design the reaction so it not only makes the protein but also gives it a membrane-like environment to fold into correctly. One of the main challenges with membrane proteins is that they tend to misfold, aggregate, or precipitate because their hydrophobic regions do not stay stable in plain aqueous solution. To deal with that, I would test conditions that include detergents, liposomes, or nanodiscs so the protein has somewhere to insert during or right after translation. I would also optimize variables like magnesium concentration, temperature, reaction time, and DNA concentration, since these can strongly affect yield and folding quality. On top of that, I would check expression using something like SDS-PAGE or a tagged reporter, then compare solubility and activity across conditions.

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.

• Low protein yield in a cell-free reaction can arise from numerous sources, but three common causes are the following. First, degradation of the DNA template or mRNA transcript by nucleases present in the extract can sharply reduce output. This can be addressed by switching from linear PCR products to circular plasmid DNA, adding RNase inhibitors, and verifying template integrity by gel electrophoresis before use. Second, depletion of energy substrates or accumulation of inhibitory byproducts such as inorganic phosphate can stall translation mid-reaction. This is best addressed by switching to a more robust energy regeneration system (e.g., PEP/pyruvate kinase), adjusting the starting concentrations of NTPs and amino acids, and running time course sampling to identify when the reaction plateaus. Third, poor translation efficiency caused by suboptimal codon usage, weak ribosome binding site strength, or mRNA secondary structure near the start codon can limit ribosome loading. This can be addressed by codon optimizing the gene for the extract source, redesigning the 5’ UTR and RBS using established calculators, and introducing silent mutations to disrupt inhibitory secondary structures near the translation initiation site.

Homework questions from Kate Adamala

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

1. Pick a function and describe it.
   1. What would your synthetic cell do? What is the input and what is the output?
      I would design a synthetic minimal cell that acts as a biosensor for plant stress-related molecules, such as reactive oxygen species (ROS). The synthetic cell would detect ROS and respond by producing a bioluminescence as a direct output. Input: ROS or a plant stress-associated molecule. Output: light produced by the synthetic minimal cell itself.
   2. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
      Yes, this could technically be done in a cell-free Tx/Tl system without encapsulation. However, encapsulation adds structure and allows better control over diffusion, stability, and modular design.
   3. Could this function be realized by genetically modified natural cell?
      Yes, this could be achieved using a genetically engineered bacterium or yeast cell that senses ROS and produces light. However, using a synthetic minimal cell avoids the complexity of maintaining a living system and allows more precise control over the components.
   4. Describe the desired outcome of your synthetic cell operation.
      The desired outcome is a controllable, cell-like biosensor that produces a visible light signal in response to plant stress molecules, which could be used in vitro to study stress signaling or to prototype synthetic biology circuits for future applications like glowing plants.
2. Design all components that would need to be part of your synthetic cell.
   1. What would be the membrane made of?
      The membrane could be made from phospholipids, such as a liposome-based membrane, possibly with cholesterol added to improve stability.
   2. What would you encapsulate inside? Enzymes, small molecules.
      A cell-free Tx/Tl system, a DNA construct containing a ROS-responsive regulatory element linked to a luciferase (Luz) reporter gene, amino acids, nucleotides, salts, cofactors, an energy regeneration system to support protein production, and the luciferin substrate 3-hydroxyhispidin so that bioluminescence occurs immediately upon luciferase expression.
   3. 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)
      Bacterial (E. coli-based) is sufficient for this design because the goal is to sense a small molecule-related stress signal. ROS-responsive genetic elements function well in bacterial Tx/Tl systems, and the fungal luciferase does not require eukaryotic post-translational modifications to fold and function.
   4. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)
      The synthetic cell would communicate with the environment by allowing the input molecule, such as ROS or a small diffusible stress-related compound, to cross the membrane if it is membrane-permeable. The output would be light generated inside the synthetic cell itself, so no additional membrane channel would be needed for signal release.
3. Experimental details
   1. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)
      
      • Lipids: POPC and cholesterol could be used to form a stable liposome membrane.
      • Genes: I would include the luz gene encoding fungal luciferase under the control of an ROS-responsive bacterial regulatory element, such as an OxyR-regulated promoter like PahpC or PkatG. If I wanted the system to make its own substrate instead of adding it directly, I could also include h3h and hisps, which are part of the fungal bioluminescence pathway upstream of luz.
      • Tx/Tl system: an E. coli-based cell-free transcription/translation system.
   2. How will you measure the function of your system?
      I would measure the function of the system by monitoring light output with a plate reader or luminometer after adding the ROS input. The main readout would be bioluminescence intensity over time, comparing reactions with and without ROS to confirm that the synthetic cell is responding specifically to the stress signal. I could also compare different ROS concentrations to see how sensitive the system is.

Homework questions 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.
  • A freeze-dried cell-free dyeing patch integrated into fabric that produces natural pigments on demand when activated by moisture, enabling wearers to “grow” custom patterns and colors into their clothing without synthetic dyes or industrial processing.
• How will the idea work, in more detail? Write 3-4 sentences or more.
  • The idea would work by embedding small freeze-dried cell-free reaction patches into specific regions of the clothing, either during fabrication or as add-on design modules. These patches would contain the transcription/translation machinery, DNA templates encoding pigment producing enzymes, and the chemical precursors needed to generate color when the system is activated by water or a moisture containing spray. Once activated, the cell-free system would begin producing the enzymes, which would then convert the stored precursors into visible natural pigments directly within the patch or fabric region. This would allow the wearer to trigger color formation only when desired, creating custom patterns or designs on demand without relying on conventional dye baths, harsh chemical processing, or synthetic dyes.
• What societal challenge or market need will this address?
  • This concept addresses the environmental and sustainability problems associated with traditional textile dyeing, which often requires large amounts of water, harsh chemicals, and energy intensive industrial processing. It also responds to a growing market interest in sustainable fashion, customizable clothing, and ethically produced fashion. By allowing pigments to be generated on demand directly within the fabric, this system could reduce waste, lower the need for synthetic dyes, and give consumers a more personalized and low impact way to design or refresh their clothing.
• How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?
  • I would address these limitations by designing the dyeing patches as sealed, modular units protected by a hydrophobic semi-permeable barrier, such as a thin silicone or wax coating, that blocks accidental activation from sweat, humidity, or rain. The patches would only activate when the wearer applies a specific spray containing both water and a co-activator, such as a mild surfactant or chemical trigger not normally present in sweat or laundry conditions. After pigment production, a fixative or heat setting step could be used to lock the color into the fabric and render the spent patch inactive, allowing the garment to be worn and washed more normally afterward. This would make the system more practical while preserving its customizable, on-demand function.

Homework questions 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/ .

1. 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)

• Growing plants in space is a major challenge because microgravity, radiation, and limited resources can disrupt plant growth and trigger stress responses that are difficult to monitor in real time. This is significant because plants are essential for food production, oxygen generation, and long-term human survival during missions to the Moon and Mars. Current monitoring methods are limited in space, so there is a need for simple, portable tools. Cell-free systems like BioBits®, which can produce detectable proteins without living cells, offer a promising approach to developing on-demand biosensors to detect plant stress in space.

2. 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)

• Reactive oxygen species (ROS) responsive genetic elements, such as OxyR-regulated promoters, to detect oxidative stress signals in plants exposed to microgravity and radiation conditions.

3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

• Reactive oxygen species (ROS) are common indicators of plant stress and tend to increase when plants are exposed to challenging conditions such as radiation, altered gravity, and other environmental stresses associated with spaceflight. Because oxidative stress can occur before visible damage appears, ROS responsive genetic elements provide a useful early molecular target for monitoring plant health in space. By focusing on these stress response pathways, this proposal aims to detect when plants are beginning to experience harmful conditions, which could help support more reliable plant growth systems for long duration space missions.

4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

• My hypothesis is that a freeze-dried, BioBits® based cell-free biosensor containing a ROS responsive genetic element linked to a fluorescent reporter gene can detect oxidative stress in space grown plants earlier and more reliably than visual inspection alone. The reasoning behind this is that ROS accumulation is one of the earliest molecular responses to environmental stress in plants, often occurring before any visible symptoms appear. By coupling a ROS sensitive promoter to a fluorescent reporter visualized with the P51 Molecular Fluorescence Viewer, the biosensor would produce a measurable signal when exposed to ROS released by stressed plant tissue. Because BioBits® reactions are shelf stable, lightweight, and require no living cells, this approach is well suited to the resource limited environment of spaceflight and could provide astronauts with a simple, portable tool for monitoring crop health during long duration missions.

5. 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)

• I would test extracts collected from healthy plants and from plants exposed to a space relevant oxidative stress condition, along with a no sample negative control and a positive control containing a known ROS source. Each sample would be added to freeze-dried BioBits® reactions containing a ROS responsive promoter linked to a fluorescent reporter, then fluorescence would be measured with the P51 viewer and compared across conditions. If needed, miniPCR® could be used to amplify the DNA template before the BioBits reaction. The main data collected would be fluorescence intensity or visible signal strength for each treatment.