Week 9 Lab: Cell-Free Systems

Special Note: As a Committed Listener without lab access, documentation within my page is for purely acknowledgement purposes. There were no explicit instructions for Committed Listeners to submit lab work for this week, and with that, I reviewed the material.

Homework, shown on the page of Week 9 is reposted here to ease interpretation and documentation.

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.

Cell free expression allows teams to conduct biomanufacturing without living cells and operate beyond the constraints of productions in living systems.

Two cases where cell-free expression is more beneficial than cell production are where:

A) biomanufacturing would kill the cells B) teams have a desire to rapidly prototype biomanufacturing workflows after computational modeling

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

The main components of cell free expression are as follows:

Cell lysate/mix: These provide the vital translation components, in addiiton to ribosomes, other enzymbes, and tRNAs Genomic Template: These are needed to encode and develop the protein of interest Salts: Ioinic condition maintenance Buffer: Maintaining pH Cofactors/additives: Promoting enzymatic activity Amino Acids: These supply building blocks for translation Energy System: These supply energy to power transcription and translation

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.

Energy provision generation is critical to sustain reactions. Supplying molecular energy packs that can regenerate ATP during a reaction can assist this.

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

Eukaryotic and prokaryotic cell-free expression systems have their own unqiue advanges. Prokaryotic cell-free expression tends to be great for rapid prototyping and is robust. Eukaryotic systems can have advantages with complex products by which post-translational modification may be desired.

I’d possibly consider developing flourescent proteins in prokaryotic systems while focusing on antibodies with eukarytic systems.

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.

A) My initial guess would be to examine setups by which I can utilize detergents and or membrane disaggrgating components and trial setups. B) Challenges come from aggregation of membrane proteins, insolubility of components, and reduced yields. I would possibly consider different spatial component arrangements, release modalities, temperature changes, and experiment with amounts of disaggrgating components.

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.

Three possible reasons could be a poor template, poor environmental considtions, and or lack of energy. Troubleshooting respectively would look like: attempting template optimization, environmental optimization, and trialing suppying more energy sources for the reaction. Each of these would be systematically trialed.