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 worrying about a cell membrane or the knock-on impacts of every modification before its more

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

• lysate: reads the DNA for protein synthesis • sequence: code for the expression construct • nuclease free water: the environment for the reaction • buffers: provides the pH, minerals, etc that are needed for the environment to be right for the reaction to occur • energy sources from enzymes and substrates: necessary to fuel the transcription and translation for the protein synthesis

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.

Usually cells have components that can continuously convert energy sources from their environment into ATP, which cell-free systems are not built to do as well. You could build a system of indirect energy sources that get triggered by stages of reactions, creating a more sustained energy supply.

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

The two cell types have a completely different structure, and the way that DNA is folded in these cells is completely different. The protein transcription would be very different, any folding tags, etc.

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.

Not enough energy source (modify the master mix), the system hasn’t run for long enough (incubate for longer), or maybe the sequence was incorrectly optimized for the lysate system in the protocol (adjust the organism its optimized for) .

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?

It would modify siliffin proteins from diatoms. the input is modified siliffin and the output would be a potential spectral shift between cell free samples

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?

Potentially, but the silica cell membrane of the diatoms make them very difficult to open for modification like gibson assembly without killing the 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.