https://pages.htgaa.org/2026a/louisa-zhu/homework/week-09-hw-cell-free-systems/index.htmlPart 1: General Homework Questions Cell’s survival is the priority. In CFPS, the “cell” is broken open, leaving only the machinery. The advantages include more direct access (adding non-canonical AA, detergents, chaperones) or an open system to monitor the reaction in real-time and adjust variables. One case where this is more beneificial would be in toxic proteins; if the protein kills a living host, it can still be produced in a cell-free system because there’s no “life” to extinguish. Another case could be for rapid prototyping, where CFPS allows for cycles in hours rather than the days required for usual cell transformation and growth. Major components: crude extract provides things like ribosomes, RNA polymerase, translation factors, the DNA template encodes the protein, the energy mix provides energy, amino acids provide building blocks for proteins and cofactors/salts provide ions that help stability and enzyme activity. Energy provision regeneration is critical because translation is energy-intensive. Every peptide bond requires the hydrolysis of multiple high-energy phosphate bonds. Without regeneration, ATP levels plummet in minutes, and the accumulation of inorganic phosphate inhibits the reaction. Something possible for continuous supply would be to use Secondary Energy Source, such as the Creatine Phosphate/Creatine Kinase system. Creatine kinase transfers a phosphate group from creatine phosphate back to ADP, maintaining a steady-state concentration of ATP throughout the batch reaction. Prokaryotic: High yield, fast and cheap. A protein that might be produced is GFP, that is simple and doesn’t require complex folding or glycolysations. Eukaryotic systems provide lower yield but are capable of complex post-translational modifications like glycosylation and proper disulfide bond formation. A protein that might be produced in one of these systems is human insulin, which requires specific folding and bridges that the eukaryotic machinery handles better. When encountering low protein yields in a cell-free system, the first step is often to investigate template stability. In many extracts, endogenous nucleases are present that can rapidly degrade linear DNA templates. A common troubleshooting strategy is to switch to a circular plasmid or supplement the reaction with nuclease inhibitors like Gam protein. A second common culprit is codon bias, where the genetic sequence of the target protein utilizes codons that are rare within the organism from which the extract was derived. This can be addressed through synonymous gene sequence optimization or by using specialized extracts supplemented with rare-target tRNAs. Finally, the concentration of magnesium ions is a critical variable that often requires a titration experiment. Because magnesium is essential for ribosome assembly but inhibitory at high concentrations, performing a series of reactions across a gradient is a standard strategy to find the “sweet spot” for a specific proteins. Part 2: Homework Questions from Kate Adamala Designing a cell-free experiment for membrane proteins introduces the unique challenge of hydrophobicity, as these proteins often aggregate or misfold without a lipid environment. To address this, one can incorporate synthetic surfactants or detergents into the reaction to keep the protein soluble during synthesis. Alternatively, a more biomimetic approach involves adding nanodiscs or liposomes directly into the cell-free mix, providing a membrane-like scaffold for the protein to insert into co-translationally. The primary advantage here is that the open nature of the system allows you to precisely control the lipid composition to optimize the stability and activity of the membrane protein without the toxicity issues often seen in living hosts.Hugoen