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.
  • One advantage includes the fact that cell-free protein synthesis gives direct control over reaction composition which means we can precisely control the exact concenctrations of factors like DNA, amino acids and modified nucleotides.
  • No cell membrane barrier means everything is immediately accessible and modifiable
  • The speed at which you can cycle designs and tests is much faster thant that if you’d have to do the usual process of cloning then transform then grow
  • You can precisely and exactly control the specific environments its in, setting the exact pH, temperature and also be able to eliminate toxic constraints.

  1. The bread and butter for cell free systems is the cell extract which includes the ribosomes that are the machinery for protein synthesis, tRNAs to deliver amino acids, translation factors and also the enzymes required for and involved in metabolism.

  2. Energy regeneration is critical in cell-free protein synthesis because ATP is rapidly consumed during transcription and translation, and unlike in living cells, there are no metabolic pathways to replenish it, causing protein synthesis to quickly stop if energy is depleted. Simply adding ATP is insufficient due to rapid consumption and accumulation of inhibitory byproducts, so regeneration systems are required to sustain reactions and improve yield. One common method is the phosphoenolpyruvate (PEP) system, in which PEP donates a phosphate group to ADP via enzymes such as pyruvate kinase present in extracts from Escherichia coli, continuously regenerating ATP and allowing protein synthesis to proceed for longer durations.

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

  • Prokaryotic cell-free systems, typically derived from Escherichia coli, are fast, cost-effective, and produce high yields, but lack the machinery for complex post-translational modifications such as glycosylation or proper disulfide bond formation.

  • In contrast, eukaryotic systems (e.g., wheat germ or rabbit reticulocyte extracts) are slower and more expensive but support proper folding and modifications required for many eukaryotic proteins.

  • For example, a simple enzyme such as Green Fluorescent Protein can be efficiently produced in a prokaryotic system because it does not require extensive post-translational modifications, making it ideal for rapid, high-yield expression. Conversely, a therapeutic protein like Insulin is better suited to a eukaryotic system, as it requires correct folding and disulfide bond formation to be biologically active.

  1. 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 optimise expression of a membrane protein in a cell-free system, I would design the experiment as a small screening setup in which the same DNA template is expressed across multiple reaction conditions while varying factors that most strongly affect membrane protein yield and solubility
  1. This could most likely be due to a low concentration of the DNA fragment thats coding for the target protein we can fxi this by increasing the DNA fragment concentration

this could also be due to an enzyme thats accidentally been released into our cell free system thats cleaving our target protein and so we can fix this by checking and ensuring no said enzymes are produced.

This could also be due to a lack of amino acid building blocks to actually synthesise the protein and so we can add a better nutrient medium to ensure this is not in limiting.

Kate Adamala’s HW

  1. My synthetic minima