Week 12 HW: Bioproduction

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Part 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.

Part 3: Designing Experiment

Properties of Proteins

  • sfGFP (Superfolder GFP): Known for its robust and rapid folding kinetics, sfGFP can fold correctly even when fused to poorly behaved proteins, making it the most reliable “readout” for initial expression levels in lysate.
  • mRFP1 (monomeric Red Fluorescent Protein): A key property is its relatively low quantum yield and slow maturation compared to newer variants; in a cell-free system, this may lead to a delayed or dim signal if the incubation ends before the chromophore fully oxidizes.
  • mKO2 (monomeric Kusabira Orange 2): This protein features excellent pH stability and rapid maturation, allowing it to maintain a bright orange signal even if the cell-free reaction becomes slightly acidic due to metabolic byproduct accumulation.
  • mTurquoise2: It is characterized by a high fluorescence quantum yield and superior photostability, making it ideal for high-sensitivity imaging, though its cyan emission is highly dependent on the correct folding of its rigid beta-barrel structure.
  • mScarlet-I: This is one of the brightest red FPs available but is notably sensitive to oxygen levels for chromophore maturation; in a 36-hour cell-free “painting” (especially if wells are sealed), limited oxygen may significantly cap its peak fluorescence.
  • Electra2: Specifically designed for rapid maturation, Electra2 is intended for real-time monitoring of translation, meaning its signal will likely peak much earlier in the 36-hour window than more stable but slower-maturing proteins like mRFP1.

Hypothesis

Protein: mScarlet-I

Reagent(s): Catalase or increased HEPES-KOH concentration

Expected Effect: By increasing the HEPES-KOH concentration or adding Catalase, we can counteract the “acid sensitivity” and oxidative stress that often occurs during a prolonged 36-hour incubation. Since mScarlet-I is highly bright but sensitive to its environment, maintaining a stable pH and reducing reactive oxygen species (ROS) from the long-term metabolic activity of the NMP-Ribose-Glucose system will prevent premature quenching and maximize the final fluorescence intensity.