Week 9 HW: Cell Free Systems

#General homework questions

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.

From “A User’s Guide to Cell-Free Protein Synthesis,” https://pmc.ncbi.nlm.nih.gov/articles/PMC6481089/

Two advantages would be 1) “elimination of reliance on living cells,”" so we don’t hve to wait for the living organism’s growth cycle an maintain their living conditions, and 2) “the ability to focus all system energy on production of the protein of interest.” Two cases where cell-free systems have benfits over in vivo methods include 1) “The production of functional antibodies and antibody fragments in vitro using CFPS has the potential to allow for simplification of the antibody production process for more rapid manufacturing,” and 2) “Metalloproteins … are difficult to produce in vivo … have the potential to enable renewable hydrogen fuel and other important biotechnological advancements.”

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

From “Cell-Free Protein Expression” https://www.neb.com/en-us/applications/protein-expression/cell-free-protein-expression

“In CFPS, a solution containing all the cellular machinery needed to direct protein synthesis (e.g., ribosomes, tRNAs, enzymes, cofactors, amino acids, etc.) is used to transcribe and translate a supplied nucleic acid template (e.g., plasmid DNA, linear DNA or mRNA).”

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.

From “Energy systems for ATP regeneration in cell-free protein synthesis reactions” https://pubmed.ncbi.nlm.nih.gov/17634594/

“Supplying energy for cell-free protein synthesis reactions is one of the biggest challenges to the success of these systems. Oftentimes, short reaction duration is attributed to an unstable energy source. "

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

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.

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.