https://pages.htgaa.org/2026a/yutong-wu/homework/week-09-hw-cell-free-systems/index.html##Part 1 1.Advantages of Cell-Free Over In Vivo Expression Cell-free protein synthesis (CFPS) removes the cell as a “black box” and allows you directly control and observe every variable in real time: pH, redox potential, ionic strength, and cofactor concentration. 2.Main Components and Their Roles Component Role Cell extract Provides ribosomes, tRNA, synthetases, chaperones, and machinery DNA/mRNA template Encodes the target protein (plasmid or linear) RNA polymerase Transcribes DNA → mRNA (T7 RNAP is most common) Amino acids Raw building blocks for translation Energy system Supplies and recycles ATP/GTP to power translation Salts and buffer Maintains pH (~7.5) and ionic strength (Mg²⁺, K⁺ critical) Additives Chaperones, detergents, etc., added based on target needs 3.Energy Provision and ATP RegenerationHugoen