The-components-of-a-cell-free-protein-synthesis-reaction-The-reaction-is-assembled-in-a.png

Lecture (4/1)

<aside> <img src="/icons/slide_green.svg" alt="/icons/slide_green.svg" width="40px" /> Lecture slides will be shared here after the class!

</aside>

<aside> <img src="/icons/video-camera_yellow.svg" alt="/icons/video-camera_yellow.svg" width="40px" /> Class Recording

</aside>

Recitation (4/2)

<aside> <img src="/icons/slide_green.svg" alt="/icons/slide_green.svg" width="40px" /> Recitation slides will be shared here after the class!

</aside>

<aside> <img src="/icons/video-camera_yellow.svg" alt="/icons/video-camera_yellow.svg" width="40px" /> Recitation Recording

</aside>

Homework Part A

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Mandatory for Committed Listeners and MIT/Harvard Students

</aside>

<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" />

</aside>

  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.

Cell-free synthesis allows precise control over reaction conditions, enabling direct manipulation of transcription and translation. It facilitates the incorporation of non-standard amino acids and eliminates cell viability constraints. This approach is particularly useful for expressing toxic proteins, as no living cells are affected, and for rapid protein production, bypassing time-consuming cell culture steps.

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

A cell-free system consists of a crude or purified extract providing the transcription and translation machinery, an energy source to sustain protein synthesis, amino acids as building blocks, cofactors and salts for enzymatic activity, and a template DNA or mRNA encoding the target protein.

  1. 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.

Energy regeneration is crucial because ATP is rapidly consumed during translation, and depletion halts protein synthesis. A common approach is using a secondary energy source such as phosphoenolpyruvate (PEP) or creatine phosphate, which regenerates ATP through enzymatic reactions, maintaining synthesis efficiency.

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

Prokaryotic systems, such as E. coli extracts, are fast and cost-effective, making them suitable for simple, soluble proteins like GFP. Eukaryotic systems, such as wheat germ or mammalian extracts, enable proper folding and post-translational modifications, making them ideal for complex proteins like antibodies.

  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.

Membrane proteins pose solubility challenges, often aggregating without a lipid environment. To optimize expression, detergents, nanodiscs, or liposomes can be added to mimic native membrane conditions. Adjusting Mg²⁺ and K⁺ concentrations can further enhance stability and yield.

  1. 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.

Low yield can result from insufficient energy supply, template degradation, or improper folding. Supplementing an ATP regeneration system can counteract energy depletion. Using RNase inhibitors prevents mRNA degradation. Adding chaperones or optimizing reaction conditions improves folding efficiency.