https://pages.htgaa.org/2026a/louisa-zhu/homework/week-12-hw-bioproduction/index.htmlPart 1: Global Pixel Art So, unfortunately I didn’t receive an email to contribute to this, and I saw the homework posting AFTER the deadline. I guess I will just have to TA next year instead! Part 2: Cell Free Synthesis E. coli Lysate BL21 (DE3) Star Lysate: Provides the essential cellular machinery, including ribosomes, tRNAs, and initiation/elongation factors, required for translation. T7 RNA Polymerase: Specifically transcribes the target DNA template into mRNA, driving high-level protein expression. Salts/BufferPotassium Glutamate: Acts as the primary salt to maintain ionic strength and provides potassium ions, which are vital for ribosomal activity. HEPES-KOH (pH 7.5): A buffering agent that stabilizes the pH of the reaction, ensuring enzymatic activity remains optimal as metabolic byproducts accumulate. Magnesium Glutamate: Supplies $Mg^{2+}$ ions, which are critical cofactors for polymerase activity and the structural stability of ribosomes. Potassium Phosphate (Monobasic/Dibasic): Works alongside HEPES to provide secondary buffering capacity and maintains inorganic phosphate levels for energy cycling. Energy / Nucleotide System Ribose & Glucose: Serve as carbon and energy sources that the lysate’s endogenous pathways use to regenerate ATP. AMP, CMP, GMP, UMP (NMPs): These monophosphate nucleotides are the raw building blocks that the system phosphorylates into NTPs for RNA synthesis. Guanine: Acts as a precursor to maintain the pool of guanosine nucleotides, which are essential for the initiation and translocation steps of translation. Translation Mix (Amino Acids)17 Amino Acid Mix / Tyrosine / Cysteine: These are the physical building blocks of the protein; Tyrosine and Cysteine are often added separately due to lower solubility or specific stability requirements. Additives & Backfill Nicotinamide: Helps stabilize and regenerate $NAD^{+}$ levels, supporting the metabolic flux required for sustained energy production.Nuclease Free Water: Acts as the solvent for the reaction, ensuring no residual enzymes degrade the DNA template or mRNA products. Comparing Master Mixes The 1-hour PEP/NTP mix is designed for speed and immediate energy, utilizing pre-formed NTPs and Phosphoenolpyruvate (PEP) as a direct, high-energy phosphate donor for rapid, short-burst reactions. In contrast, the 20-hour NMP-Ribose-Glucose mix is optimized for sustainability and cost-effectiveness, using cheaper precursors (NMPs and sugars) that the system slowly converts into energy via endogenous metabolism to support protein production over a much longer duration. While the 1-hour mix prioritizes a quick “sprint” for rapid results, the 20-hour mix facilitates a “marathon” by recycling energy through more complex biochemical pathways.Hugoen