Week 11: Bioproduction & Cloud Labs

Cell-Free Protein Synthesis | Cell-Free Reagents

Q1. Referencing the cell-free protein synthesis reaction composition (the middle box outlined in yellow on the image above, also listed below), provide a 1-2 sentence description of what each component’s role is in the cell-free reaction.

E. coli Lysate

  • BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): Provides the actual machinery of transcription and translation: ribosomes, tRNAs, elongation factors, and the T7 RNA polymerase that recognizes T7 promoters on your DNA template. The “Star” variant has a mutation (rne131) that reduces RNase E activity, so mRNA stays intact longer in the reaction.

Salts/Buffer

  • Potassium Glutamate: Provides K+ ions at a concentration (~312 mM) that mimics the E. coli cytoplasm, which ribosomes and most enzymes require for activity. Glutamate is used as the counterion instead of chloride because Cl- destabilizes proteins and inhibits translation, while glutamate stabilizes protein structure.
  • HEPES-KOH pH 7.5: Zwitterionic buffer that maintains reaction pH near 7.5 by absorbing protons released during glycolysis, nucleotide phosphorylation, and translation. Without it, the reaction would acidify over the long incubation and inactivate enzymes.
  • Magnesium Glutamate: Provides Mg2+, the essential cofactor for RNA polymerase, ribosomes, and every enzyme that handles phosphate groups (kinases, polymerases). Without sufficient Mg2+, neither transcription nor translation can proceed.
  • Potassium phosphate monobasic (KH2PO4): Supplies inorganic phosphate (Pi) used by lysate kinases to phosphorylate NMPs into NTPs, and contributes to pH buffering on the acidic side together with the dibasic form.
  • Potassium phosphate dibasic (K2HPO4): Same Pi supply role as monobasic, but contributes to buffering on the basic side; together the mono/dibasic pair forms a phosphate buffer that backs up HEPES.

Energy / Nucleotide System

  • Ribose: Feeds the pentose phosphate pathway in the lysate to generate PRPP (5-phosphoribosyl-1-pyrophosphate), the sugar-phosphate scaffold needed by salvage enzymes to make GMP from free guanine.
  • Glucose: Primary energy substrate; lysate glycolysis enzymes break it down to regenerate ATP from ADP, sustaining the energy supply throughout the long incubation.
  • AMP: Adenosine monophosphate; lysate kinases phosphorylate it stepwise (AMP → ADP → ATP) to supply ATP both as an NTP for transcription and as the universal energy currency for translation.
  • CMP: Cytidine monophosphate; phosphorylated stepwise by lysate kinases to CTP for transcription. Supplied as the cheap monophosphate instead of CTP directly.
  • GMP: Guanosine monophosphate; would normally be phosphorylated to GTP for transcription, but in this formulation it is set to 0 µM and replaced by free guanine plus ribose via the purine salvage pathway (see bonus question).
  • UMP: Uridine monophosphate; phosphorylated stepwise by lysate kinases to UTP for transcription.
  • Guanine: Free purine base, the cheapest entry point to the GTP pool; lysate enzymes combine guanine with PRPP from ribose to make GMP via salvage, then phosphorylate GMP up to GTP.

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: Combined stock of 17 of the 20 standard amino acids, which ribosomes incorporate into the growing polypeptide during translation. Running out of any single amino acid stalls protein synthesis.
  • Tyrosine pH 12: Added separately because tyrosine has poor solubility at concentrated stocks and requires basic pH to stay dissolved.
  • Cysteine: Added separately because cysteine readily oxidizes to cystine (the disulfide-bonded dimer) in solution, which cannot be incorporated into protein; keeping it as a fresh separate component maintains it in its usable reduced form.

Additives

  • Nicotinamide: Precursor for NAD+ biosynthesis; NAD+/NADH is consumed during glycolysis (specifically by GAPDH), so replenishing the precursor pool keeps glycolysis and ATP regeneration running through the long incubation.

Backfill

  • Nuclease Free Water: Brings the reaction up to its final 20 µL volume; must be nuclease-free because trace RNases or DNases would degrade the mRNA and template DNA, killing the reaction.

Q2. Describe the main differences between the 1-hour optimized PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix shown in the Google Slide above.

The 1-hour PEP-NTP mix supplies the four NTPs (ATP, GTP, CTP, UTP) directly and uses PEP plus lysate pyruvate kinase to regenerate ATP from ADP rapidly, so transcription starts immediately, but the system burns out within hours as PEP gets depleted and inhibitory pyruvate accumulates. The 20-hour NMP-Ribose-Glucose mix instead supplies cheap monophosphates (AMP, CMP, UMP) plus free guanine and ribose, which lysate kinases and salvage enzymes have to phosphorylate up to NTPs, and uses glucose feeding glycolysis as a slow but self-sustaining ATP regeneration source supported by nicotinamide-replenished NAD+. The trade-off is speed versus duration and cost: the PEP-NTP system is fast and expensive (~10x cost per reaction), while the NMP-Ribose-Glucose system has a slower start but runs productively for 20+ hours at a fraction of the cost, making it the better choice for high-throughput autonomous lab work and long-incubation experiments like the 36-hour artwork.