https://pages.htgaa.org/2026a/jason-ross/homework/week-09-hw-cell-free-systems/index.htmlHomework Part A: General and Lecturer-Specific Questions 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. Cell-free systems allow for a broader range of potential chemistries than those given to us from natural biology, expanding flexibility. Cell-free protein synthesis also allows for greater control over experimental variables because the entire protein expression construct is designed from scratch (i.e., we have the opportunity to bypass a lot of the compleity of natural cells). Cell-free expression is more beneficial than cell production if you want to rapidly protoype gene pathways and if you want an expression mechanism that’s more amenable to consistent, predictable modeling and analysis. Describe the main components of a cell-free expression system and explain the role of each component. The main components of a cell-free expression system are (based on elements described in this hyperlink 1): DNA template: Genetic code to begin Tx/Tl process Ribosomes: Assembling amino acids into polypeptides Enzymes: Catalyzing certain important chemical reactions necessary for the appropriate functioning of that cell-free expression system (ex. transcription and translation, energy generation) Amino Acids: The core chemical building blocks of the proteins the cell-free expression system will express Polymerases: Synthesizing DNA and RNA 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 regeneration is critical in cell-free systems because cell-free systems don’t consume enzymes to produce energy. They also need external energy sources to remove waste products. A workaround might be to have analogous enzymatic reactions (possibly based off shared common charges) within the cell-free system to produce energy Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. Prokaryotic cell-free expression systems allow for the colocation of transcription and translation. This might work well for proteins that need to be produced at high volume, like an industrial protease prtoein. Eukaryotic cell-free expression systems allow for more complex proteins to be built due to their nuclei. This might work well for the production of more advanced/technically complex proteins, like rabbit serum albumin. 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. In a manner similar to Shuguang Zhang ‘molecular glove’ experiment, I’d try to essentially coat and/or surround the the membrane protein with hydrophilic proteins to attract and/or absorb water in the cell-free environment, so the membrane protein can incorporate into the liposome 2. Challenges might include appropriate hydrophilic concentrations (which might be discerned via calculations or trial and error) or bonding between the hydrophilic proteins and the membrane proteins. This might be mitigated and/or the amount of error reduced through the use of computaitonal modeling and simulation tools like AlphaFold 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. Suboptimal Ribosome Function: Examine ribosome mRNA transcription processes and modify as necessary Suboptimal Transcription: Examine tRNAs for coding errors/misreads or inappropriate expression levels and modify as necessary Suboptimal External Communication (i.e., yields cannot properly exit system at desired levels): Examine and modify membrane channel functionality as necessary Supporting prompts for this section listed below:Hugoen