Week 9 HW: Cell Free Systems

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 is more flexible than in vivo expression because you can directly control the reaction conditions without keeping cells alive. It is especially useful for making toxic proteins and proteins that are hard to express in living cells, like some membrane proteins.

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

    • A cell-free system includes the extract or machinery for transcription and translation, the DNA template, amino acids, nucleotides, salts, cofactors, and an energy source. Together, these parts let the system read the gene and build the protein outside of a living cell.

  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 regeneration is important because protein synthesis uses a lot of ATP and GTP, and the reaction will stop if energy runs out. One way to maintain ATP supply is to include an energy regeneration system such as phosphoenolpyruvate or a slower source like maltodextrin.

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

    • Prokaryotic systems are faster, cheaper, and good for simple proteins, while eukaryotic systems are better for proteins that need more complex folding or modifications. For example, GFP could be made in an E. coli system, while a human receptor protein would be better in a eukaryotic system.

  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 express a membrane protein, I would add liposomes, nanodiscs, or mild detergents so the protein has a membrane-like environment while it is being made. The main problems are aggregation and misfolding, so I would test different additives and reaction conditions to improve stability and function.

  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 yield could come from poor DNA design, non-optimal reaction conditions, or the protein being unstable or aggregating. I would troubleshoot by redesigning the construct, adjusting salts and temperature, and adding folding helpers or membrane mimics if needed.

Homework question from Kate Adamala

  1. Pick a function and describe it.

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

  • My synthetic minimal cell would support coral mineralization by producing urease activity in a controlled, cell-like system. The input would be urea, and the output would be ammonia and inorganic carbon, which can help create conditions that support calcification.

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

  • Yes, the basic function could be done with cell-free Tx/Tl alone because the system only needs to express urease. But encapsulation would make it more useful as a synthetic cell by localizing the reaction and making it easier to apply as a material.

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

  • Yes, a genetically modified microbe could also express urease and help change the local chemical environment. Still, a synthetic minimal cell may be safer and easier to control because it is non-living and cannot grow or spread.

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

  • The desired outcome is that the synthetic cell produces urease when activated and helps shift the surrounding chemistry toward conditions that support coral calcification. In the long term, this could help probiotic materials support reef building on coastal or architectural surfaces.
  1. Design all components that would need to be part of your synthetic cell.

a. What would the membrane be made of?

  • The membrane would be made of phospholipids, possibly with cholesterol for extra stability. This would create a vesicle that can hold the cell-free system and function like a minimal synthetic cell.

b. What would you encapsulate inside?

  • Inside, I would encapsulate the cell-free transcription-translation system, DNA for urease expression, amino acids, nucleotides, salts, cofactors, and an energy source. I would also include any helper components needed for urease folding and activity.

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

  • I would use a bacterial cell-free system, since urease expression can be handled in a bacterial context and the design does not require mammalian regulation. A bacterial system is simpler and more practical for a first prototype.

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

  • The system would communicate with the environment by taking in urea from the surrounding water and converting it through urease activity. The products of that reaction would diffuse out and change the local chemical conditions around coral or the material surface.
  1. Experimental details

a. List all lipids and genes.

  • The main lipids would be phospholipids such as POPC, with cholesterol if needed for stability. The genes would include the urease gene cluster and any accessory genes needed for proper urease assembly and activity.

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

  • I would measure function by checking whether urease is successfully expressed and whether urea is broken down. I would also measure changes in pH, ammonia production, or other chemical signs that the system is creating conditions favorable for mineralization.

Homework question from Peter Nguyen

  1. Write a one-sentence summary pitch sentence describing your concept.

    • I propose a freeze-dried probiotic-inspired material that uses cell-free urease expression to create local chemical conditions that support coral mineralization on coastal and architectural surfaces.

  2. How will the idea work, in more detail? Write 3-4 sentences or more.

    • The material would contain freeze-dried cell-free components programmed to express urease when activated by water and supplied with urea. Once active, the system would break down urea into products that may help create conditions favorable for coral calcification. This would let a surface act not just as passive infrastructure, but as a bioactive partner in reef-building processes.

  3. What societal challenge or market need will this address?

    • This idea addresses the need for new ways to support coral reef restoration and climate-adaptive coastal design. It also responds to the growing need for architectural materials that do more than resist environmental change and instead actively contribute to ecological recovery.

  4. How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

    • Freeze-drying helps the system stay stable until it is needed, and water activation is useful because the material would only turn on in wet coastal conditions. Since cell-free reactions are temporary, the material could be designed as a replaceable or renewable layer rather than a permanent system.

Homework question from Ally Huang

  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)

    • Water safety is a major challenge in space because contamination can threaten astronaut health. A freeze-dried cell-free biosensor would be useful because it is lightweight, stable, and easy to activate when needed.

  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)

    • My target is a reporter circuit that responds to a contamination-related molecule and produces GFP. This makes hidden contamination easier to detect.

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

    • The target matters because it converts possible water contamination into a visible fluorescent signal. That would make routine testing easier in a resource-limited space environment.

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

    • My hypothesis is that a freeze-dried cell-free biosensor can act as a simple first-pass test for water contamination in space. If it gives a reliable fluorescent response, it could become a useful lightweight diagnostic tool.

  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)

    • My hypothesis is that a freeze-dried cell-free biosensor can act as a simple first-pass test for water contamination in space. If it gives a reliable fluorescent response, it could become a useful lightweight diagnostic tool.