https://pages.htgaa.org/2026b/rosalea-matta/week-03-hw-cell-free/index.htmlObjective The objective of this assignment was to design novel cell-free protein synthesis (CFPS) reaction compositions for three target proteins — sfGFP, PETase, and Reteplase — with the goal of reducing the cost per gram of functional protein. CFPS uses cell lysate containing the machinery required for transcription and translation, combined with DNA, amino acids, nucleotides, energy sources, salts, cofactors, and other components. Since individual reaction components can be adjusted independently, CFPS provides a flexible platform for optimizing protein production and cost. The three proteins were selected because they represent increasing levels of folding difficulty: sfGFP has no disulfide bonds, PETase has one disulfide bond, and Reteplase has nine disulfide bonds. A. COMPARE CELL-FREE REACTION COMPOSITIONS (20 minutes) Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive? Potassium glutamate (K-Glu) and magnesium glutamate (Mg-Glu) both contribute to the ionic environment required for efficient transcription and translation. However, they do not play identical roles. K-Glu primarily helps establish the overall ionic strength of the reaction, while Mg²⁺ has a more sensitive role in processes involving nucleic acids, ribosomes, and ATP/NTP interactions. Therefore, the concentrations of K-Glu and Mg-Glu differ between optimized formulations because the two ions affect the CFPS system differently. Mg-Glu is generally the more sensitive parameter, meaning relatively small changes in Mg²⁺ concentration can significantly affect reaction performance.Hugoen