https://pages.htgaa.org/2026a/yutong-wu/labs/week-11-hw-cloud-lab/index.htmlPart B — Cell-Free Protein Synthesis B1. Role of each reagent (20 h NMP-Ribose-Glucose mix) Component Role in the reaction BL21 (DE3) Star lysate Source of ribosomes, tRNAs, aminoacyl-tRNA synthetases, and other translation machinery. (DE3) carries T7 RNAP for transcription; Star = reduced RNase E -> mRNA more stable. Potassium glutamate Dominant monovalent cation, mimics cytoplasmic ionic environment, stabilizes ribosome conformation. Glutamate (vs Cl-) doesn’t inhibit translation. HEPES-KOH pH 7.5 Zwitterionic buffer holds pH near physiological — keeps T7 RNAP and ribosomes active. Magnesium glutamate Mg 2+ cofactor for ribosome assembly, T7 RNAP, and aminoacyl-tRNA synthetases. Concentration is highly tunable — too low halts translation, too high promotes misincorporation. K-phosphate mono/dibasic Secondary pH buffer + phosphate pool for nucleotide kinase reactions (NMP -> NDP -> NTP). Ribose Feeds the salvage pathway: ribokinase -> ribose-5-P -> PRPP, which combines with free bases to form NMPs. Also a slow-burning energy substrate. Glucose Carbon source for glycolysis -> continuous ATP regeneration (sustained energy, unlike PEP which burns fast). AMP / CMP / UMP NMP precursors. Cellular kinases phosphorylate them to NTPs in situ -> slower ramp than feeding NTPs, but cheaper and less product-inhibition. GMP (0 mM here) Omitted in the 20 h mix; GTP is generated via the Guanine salvage path instead (see bonus). Guanine Substrate for HGPRT: Guanine + PRPP -> GMP + PPi. Cheaper than buying GMP directly. 17 amino acid mix Bulk substrate pool for translation (all proteinogenic AAs except Tyr and Cys, which need special handling). Tyrosine (pH 12) Added separately — Tyr has very low solubility at neutral pH and must be kept in alkaline solution until dilution. Cysteine Added separately — readily oxidizes to cystine (forms disulfides). Kept in its own tube to avoid inactivation before reaction start. Nicotinamide NAD+ precursor + inhibitor of NAD-degrading enzymes (NADases) in the lysate -> preserves the redox cofactor pool over the long incubation. Nuclease-free water Backfill to final volume; nuclease-free to protect the DNA template and mRNA. B2. PEP-NTP (1 h) vs NMP-Ribose-Glucose (20 h) The PEP-NTP mix feeds the reaction with finished NTPs and uses phosphoenolpyruvate as a high-energy ATP regenerator — fast, intense protein synthesis, but PEP is depleted within ~1 hour and the system burns out. The NMP-Ribose-Glucose mix instead supplies precursors (NMPs + ribose for PRPP, glucose for glycolytic ATP) and lets the lysate’s own kinases and salvage enzymes assemble NTPs on demand, giving a slower but sustained ramp that lasts 20+ hours. Cost-per-reaction is also much lower because cheap precursors replace expensive NTPs and PEP.Hugoen