https://pages.htgaa.org/2026a/jessee-svoboda/homework/week-09-hw-cell-free-systems/index.htmlContents General homework questions Homework questions from Kate Adamala Homework questions from Peter Nguyen Homework questions from Ally Huang 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. Cell-free protein synthesis avoids the requirements of a cold chain for shipping or storage, and it also can simplify complex living systems by instead adding in specific and known amounts of reagents (enzymes, nucleotides, amino acids, etc.). It is more beneficial than cell production in situations like biosensing in remote environments (infectious disease detection in remote or under-resourced locations) and biomanufacturing of toxic products (like some pharmaceuticals) because production won’t stop due to cell death. Describe the main components of a cell-free expression system and explain the role of each component. template nucleic acid: DNA or RNA encoding the gene of interest for protein cell lysate (collection of active components, including the following - or the purified components could be added individually) tRNA: recognizes RNA codons and adds new amino acids onto a protein chain during translation polymerase: makes nucleic acids (DNA or RNA) nucleotides: used by polymerase to make nucleic acids buffer: maintains reaction pH to optimal level for enzyme function other enzymes and cofactors, depending on the goal of the system (sometimes these are included through a cell lysate) amino acids and ribosomes, if protein production is the goal 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 are essentially a series of chemical reactions (biological in nature, but still chemistry), which means that activation energy is required for some reactions. Energy provision regeneration is critical to ensure that the reactions continue to happen instead of stalling out early. Specifically, this is important in protein expression because translation is energetically expensive (requires ATP to attach amino acids to tRNAs). Cells generate ATP through a collection of metabolic processes; a cell-free system needs to be designed to ensure it has a way to generate ATP. One potential method is adding NAD and CoA to generate ATP from pyruvate without needing any additional enzymes. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. Prokaryotic systems are simpler than eukaryotic systems. Eukaryotic systems might have more components, especially for production of functional proteins, for chaperones or post-translational modifications. A prokaryotic system might be good to produce antimicrobial peptides because you don’t need to worry about the product killing the host. A eukaryotic system might be better at producing functional antibodies because antibodies are eukaryotic proteins and therefore might be more functional in a eukaryotic system. 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 membrane protein is difficult to produce in a cell-free system because it likely has a hydrophobic area and a hydrophilic area because it is natively located within a membrane. This means that it is unlikely to be folded into the correct structure without a hydrophilic space for the hydrophilic component of the protein. To optimize the expression of a membrane protein in a cell-free experiment, you would need to stabilize it, for example, by providing liposomes or membrane vesicles in which the membrane proteins could localize for correct folding. 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 for a low protein yield in a cell-free system is insufficient transcription, insufficient translation, or a badly designed DNA template. Insufficient transcription could be due to not adding enough nucleotides into the reaction. This could be tested by adding an mRNA template into the reaction to see if this solves it. Insufficient translation could be due inactive tRNA, inactive ribosomes, or not enough amino acids. This could be tested by spiking more of those individual, purified components (or fresh cell lysate) into the reaction - it’s possible one of those has been degraded. A badly designed DNA template might have a promoter that isn’t recognized by the polymerase provided in the cell-free system; this could be tested with a control reaction that includes a DNA template known to work in this established system. Reference Hunt, AC; Rasor, BJ; Seki, K; et al. Cell-Free Gene Expression: Methods and Applications. 2024. ACS Chemical Reviews 125(1): 91-149. DOI: 10.1021/acs.chemrev.4c00116 Homework questions from Kate Adamala Design an example of a useful synthetic minimal cell as follows:Hugoen