https://pages.htgaa.org/2026b/ali-shirazi/week-03-hw-cell-free/index.htmlA. COMPARE CELL-FREE REACTION COMPOSITIONS Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive? The best salt concentrations are not fixed numbers. They depend on what else is in the reaction. Magnesium is the clearest example: NTPs, NMPs, phosphate and amino acids all bind Mg²⁺, so a recipe that carries more of these needs more magnesium to leave enough free Mg²⁺ for the ribosomes and T7 RNA polymerase. Potassium and glutamate mostly set the ionic strength, so a formulation that already contains many charged components can get away with less added K-glutamate. On top of that, every lysate batch and every target protein has its own tolerance, which is why published recipes land on different values.Magnesium is the more sensitive of the two. Its optimum is sharp, and a change of only 1 to 2mM can noticeably reduce yield. Too little stalls transcription and translation, and too much inhibits them and can precipitate with phosphate. Potassium has a broad plateau and tolerates changes of tens of mM. For that reason Mg is normally titrated in small steps of about 1 mM, while K is scanned more coarsely.Hugoen