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.
   1. It allows for scientists to directly control reaction conditions such as ion concentrations, cofactors), as well as rapid prototyping without worrying about cell viability, and easy addition of non-natural amino acids or components.
   2. Two scenarios would be production of toxic proteins that would kill host cells, and rapid screening of protein variants without needing cloning.
2. Describe the main components of a cell-free expression system and explain the role of each component.
   • Cell Extract: Contains ribosomes, tRNAs, enzymes for transcription/translation.
   • DNA/RNA Template: Encodes the target protein.
   • Energy Source: Provides ATP/GTP for protein synthesis.
   • Amino Acids: Building blocks for 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.
   1. It is critical as protein synthesis consumes ATP rapidly, and without an energy source the reaction will stop. You can use a phosphate donor like creatine phosphate with catalyst creatine kinase to recycle ADP that is produced during protein synthesis to ATP continuously.
4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
   1. Prokaryotic systems (DNA floats in cell) such as E. Coli extracts are faster and cheaper, and ideal for simple proteins such as GFP. Eukaryotic systems (DNA in nucleus) support more complex folding or post translation modifications and are better suited for proteins like human antibodies.
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.
   1. By choosing a eukaryotic system which can handle more complex proteins such as membrane proteins. We can add lipid like components like liposomes and detergents to mimic the lipid bilayer-like environment. Adjust temperature, concentrations, and salts for ideal conditions for protein folding and stability. Use fluorescent tags to monitor protein expression.
   2. Challenges include the fact that membrane proteins require proper folding and insertion into lipid bilayers, which can be addressed by adding components to the system that mimic membranes, and optimizing concentrations to maintain solubility without denaturing the protein.
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.
   1. Possible reasons include:
      • Quality of template DNA is not good, DNA has degraded. Solution: use freshly purified or higher-quality DNA.
      • Insufficient Energy Supply and ATP depletion. Solution: add energy regeneration systems such as creatine phosphate.
      • Incorrect Reaction Conditions with not optimal ions or cofactors. Solution: optimize Mg, K concentrations and pH.