https://pages.htgaa.org/2026b/karol-eliana-cuellar-narvaez/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? Potassium glutamate sits around 130 mM in standard formulations and as high as 362 mM in cost-optimised ones; magnesium glutamate sits at 8–12 mM in almost all of them. The order-of-magnitude gap exists because the two ions do different jobs. Potassium sets ionic strength and osmolarity, reproducing the 100–200 mM potassium pool of the E. coli cytoplasm that ribosome conformation and tRNA binding evolved in. Its optimum is broad: tens of millimolar in either direction change yield gradually. Magnesium is catalytic and structural. It neutralises the phosphate backbone of RNA, is required for association of the 30S and 50S ribosomal subunits, for T7 RNA polymerase catalysis, and for aminoacyl-tRNA binding at the ribosome. Its optimum is narrow and sharply peaked. Magnesium is decisively the more sensitive of the two. Below its optimum, ribosomal subunits dissociate and transcription stalls; above it, the ribosome is over-stabilised, translational fidelity drops, and mRNA secondary structure is stabilised against unwinding. A few millimolar either way collapses the reaction. This also explains why magnesium varies between published compositions while potassium does not vary for the same reason. Two causes. First, the lysate itself carries magnesium, and the amount differs batch to batch. Second, several reaction components chelate Mg²⁺ — nucleotide triphosphates, phosphoenolpyruvate and inorganic phosphate all bind it — so a PEP + NTP formulation needs more added magnesium than an NMP formulation with no PEP. Olsen et al. (2026) make this explicit, re-determining the optimum magnesium and phosphate concentrations for every lysate batch and every lysate concentration. A note on the counter-ion: both salts are supplied as glutamate rather than chloride or acetate, because glutamate is the dominant physiological anion in E. coli and supports higher yields.Hugoen