<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> Mandatory for Committed Listeners and MIT/Harvard Students
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<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links:
https://dnadots.minipcr.com/wp-content/uploads/2019/09/DNAdots-Cell-Free-Tech-final_qnoa.pdf
https://pubs.acs.org/doi/10.1021/acssynbio.3c00733
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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 protein synthesis or CFPS does not require the culture and maintenance of the host organism which are occasionally pathogenic. We can just produce the target protein without much hassle.
CFPS is a better option than cell-based protein synthesis to produce viral proteins and other proteins from pathogens as it does not require the culture of viruses or those pathogens.
Describe the main components of a cell-free expression system and explain the role of each component.
Template DNA: Which codes for the protein.
RNA polymerase: Does the transcription to produce RNA from DNA template.
dNTPs: For transcription.
Amino acids: For translation.
Cofactors: For proper enzyme activity and efficiency.
Cell lysates: All necessary components to facilitate the protein production.
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.
Cell-free systems lack the natural metabolic processes like normal cells, therefore they require ATP supplement. Without a steady supply of ATP and other energy molecules, essential biochemical reactions like transcription, translation, protein folding, and enzyme activities rapidly stop.
One common method to ensure continuous ATP supply in a cell-free system is using an energy regeneration system based on phosphoenolpyruvate (PEP). To obtain these, we need to add phosphoenolpyruvate (PEP) and pyruvate kinase enzyme to the cell-free mixture. It will facilitate the reaction: PEP + ADP = Pyruvate + ATP, therefore ATP generation continues.
Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
Prokaryotic cell-free expression systems do not require to facilitate with mRNA splicing and post-translational modifications, but eukaryotic cell-free expression systems require both.
GFP production in prokaryotic cell-free system, as it doesn't require post-translational modifications to function, and is easy to detect via fluorescence.
Collagen production in eukaryotic cell-free system, as the necessary protein modifications can only be done in eukaryotic setup.