Homework Part A

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.

In a cell-free system, you don’t need to keep cells alive. So you can skip all the media broth, careful temperature control, etc. Since there are no delicate cells to keep alive, you can freeze the reagents, remove all the water from them, etc., and not worry about damaging the cells.
Lower biosafety risks, because there are no living cells that can spread or infect anything.

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

For the case of protein manufacturing from DNA, you need all the components for transcription and translation. So you’d need ribosomes, RNA nucleotides for the transcription, a lot of amino acids for the translation, plus all the supporting molecules. And probably a source of energy.

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.

In a cell-based system, the cell’s normal metabolic processes provides energy for any pathways you want to implement.

I asked ChatGPT for this. It sounds like you need some chemical that will give phosphate to ADP to “recharge” it into ATP, such as creatine phosphate.

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

I think it’s the same difference as whether in a cell-based system, you would implement your process in E. coli (prokaryotic) or yeast (eukaryotic). Prokaryotic systems are simpler, but you’re limited to processes that can be done with bacterial RNA polymerase and ribosomes.

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.

I guess the challenge is that membrane proteins normally work by seeking out and embedding themselves in the membrane, and might require the lipid layer of the membrane to work properly. But in a cell-free system, there is no membrane at all.

ChatGPT suggests detergents, which might work, because membrane proteins usually expect an environment that’s half-hydrophilic (the cytoplasm) and half-hydrophobic (the membrane), and detergents are precisely molecules that are a mix of both.

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. Try adding more of the phosphate donors as mentioned above.
Missing some component that’s normally produced by the cell. I guess you could try the same reaction in a cell and see if it works any better.
Missing a promoter that’s normally produced inside the cell. I guess you could also try the reaction in a cell and see if it works better?

Homework Part B: Individual Final Project Report