Week 11 HW: BIOPRODUCTION & CLOUD LABS

A. Master Mix Component Analysis

  1. E. coli Lysate

BL21 (DE3) Star Lysate: Provides the cellular machinery for transcription and translation, including ribosomes and T7 RNA polymerase. The “Star” mutation helps reduce mRNA degradation.

  1. Salts / Buffer

Potassium Glutamate: Maintains ionic strength and supports ribosome activity. HEPES-KOH pH 7.5: Keeps the reaction at a stable pH. Magnesium Glutamate: Supplies magnesium ions needed for ribosomes and enzymes. Potassium Phosphate: Acts as a buffer and supports ATP regeneration.

  1. Energy / Nucleotide System

Ribose & Glucose: Provide energy for ATP and GTP regeneration. AMP, CMP, GMP, UMP: Building blocks used to make RNA nucleotides for transcription. Guanine: Helps regenerate GTP through the salvage pathway.

  1. Translation Mix (Amino Acids)

17 Amino Acid Mix: Supplies amino acids for protein synthesis. Tyrosine: Added separately because it dissolves poorly. Cysteine: Added separately because it is reactive and easily oxidized.

  1. Additives & Backfill

Nicotinamide: Supports metabolic reactions by helping maintain NAD+/NADH cofactors. Nuclease Free Water: Brings the reaction to the correct volume without introducing nucleases.

–

B. Master Mix Component Analysis

The 1-hour PEP-NTP mix is designed for fast protein production using high-energy PEP and ready-made NTPs. The 20-hour NMP-Ribose-Glucose mix is designed for long-term production, using cheaper precursors and the lysate’s metabolism to slowly regenerate energy and nucleotides over time.

–

C. Planning the Global Experiment | Cell-Free Master Mix Design

Fluorescent Protein Properties

  • sfGFP: Folds quickly and reliably, so it gives strong fluorescence in many cell-free conditions.

  • mRFP1: Matures slowly, so fluorescence appears later than protein production.

  • mKO2: Very bright orange protein, but repeated reads may reduce signal through photobleaching.

  • mTurquoise2: Has high brightness, so it can be detected even at low protein yield.

  • mScarlet_I: Very bright red protein, but needs oxygen for chromophore maturation.

  • Electra2: Designed for fast maturation and stability, so it can track protein production quickly.

Hypothesis

  • Protein: mRFP1
  • Reagent adjustment: Increase magnesium glutamate slightly.
  • Hypothesis: Increasing magnesium glutamate will improve ribosome activity and translation efficiency, producing more mRFP1 over the 36-hour incubation. Because mRFP1 matures slowly, higher total protein production should lead to stronger final fluorescence after enough maturation time.