Week 9: 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.

   Because cell-free methods exist without the limitations of maintaining a living cell, they offer greater flexibility in exploring the details of protein synthesis without

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

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.

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

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.

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 Questions 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?

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:

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

   A ‘bruising’ paint that reacts to pressure, creating a lasting impression on a house as it is used more routinely.

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

   It would work by creating a biofilm ‘paint’ with pressure-reactive sequences from organisms like mimosa plants. Alternatively the biofilm could use thermoreactive organisms, marked with florescent protein to signal when the temperature is around the range of human skin.

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

   This starts a conversation around permanence in a space, and how buildings are seen as more temporary than the people as we move away from being able to afford to own our homes and instead move through rental properties, where our spaces are more defined through the objects than a sense of connection to the walls around us.

   In a healthcare sense, this could be useful to highlight surfaces that have been touched, potentially helping with sanitation for sterile rooms if we enter another large-scale health event.

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

   Walls are not often washed outside of bathrooms and kitchens perhaps, but our hands have moisture, but probably not enough to introduce water into the cell-free system. Pressure on the wall could compress and break micro-scale water cells that are suspended in the biofilm, causing a localized water exposure for the biopigment to react with.

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)

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)

   There could be a cell-free system that reacts to

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)

3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

4. 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)