Week 11 HW: Bioproduction & Cloud Labs

Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork

Part B: Cell-Free Protein Synthesis | Cell-Free Reagents

  1. Each component’s role is in the cell-free reaction.
  • E. coli Lysate (BL21 (DE3) Star): Provides the essential biological “hardware,” including ribosomes, tRNAs, and various translation factors; it specifically includes T7 RNA Polymerase to drive high-level transcription from T7 promoters.

Salts and Buffers:

    • Potassium Glutamate: Acts as the primary potassium source and a major intracellular salt, which is critical for maintaining proper osmotic pressure and supporting protein-DNA interactions during transcription and translation.
    • HEPES-KOH pH 7.5: Serves as a chemical buffering agent to maintain a stable physiological pH throughout the reaction, preventing acidification from metabolic byproducts.
    • Magnesium Glutamate: Provides essential $Mg^{2+}$ ions that act as necessary cofactors for ribosome assembly and the enzymatic activity of polymerases.
    • Potassium Phosphate (Monobasic & Dibasic): Maintains phosphate homeostasis and contributes to pH stability, while also providing inorganic phosphate for nucleotide recycling.

Energy and Nucleotide System:

    • Ribose and Glucose: Serve as secondary energy substrates that can be metabolized by residual glycolytic enzymes in the lysate to regenerate ATP sustainably.
    • AMP, CMP, GMP, UMP, and Guanine: These act as the fundamental precursors (nucleotides and bases) for mRNA synthesis during transcription and are necessary components for maintaining the energy charge of the system.

Translation Mix (Amino Acids):

    • 17 Amino Acid Mix, Tyrosine, and Cysteine: Provide the raw building blocks required for the ribosome to assemble the polypeptide chain; Tyrosine and Cysteine are often added separately due to their lower solubility at neutral pH.

Additives and Backfill:

    • Nicotinamide: Often acts as a cofactor or stabilizer to inhibit the degradation of essential metabolic intermediates like NAD+, thereby extending the reaction’s metabolic activity.
    • Nuclease Free Water: Used to adjust the final volume (backfill) of the reaction while ensuring no contaminating enzymes degrade the DNA template or mRNA products.
  1. 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. (2-3 sentences)

The 1-hour PEP-NTP mix is designed for high-speed, short-term bursts of protein synthesis by providing direct, high-energy building blocks like Nucleoside Triphosphates (NTPs) and a potent phosphagen (PEP) for immediate ATP regeneration. In contrast, the 20-hour NMP-Ribose-Glucose mix is optimized for long-term production and cost-efficiency; it utilizes cheaper precursors (Nucleoside Monophosphates) and a dual-sugar metabolic pathway (Ribose/Glucose) to regenerate energy slowly and steadily over several hours, preventing the rapid accumulation of inhibitory byproducts.

  1. Bonus question: How can transcription occur if GMP is not included but Guanine is?

Transcription can occur even if GMP is not explicitly included because the E. coli lysate contains residual metabolic enzymes (such as Phosphoribosyltransferases) that can utilize Guanine as a substrate. Through a “salvage pathway,” the system attaches a ribose-5-phosphate to the Guanine base to synthesize GMP. Once GMP is formed, kinases in the lysate further phosphorylate it into GDP and finally GTP, which is the actual nucleotide required by the RNA Polymerase to build the mRNA chain.

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

  1. Given the 6 fluorescent proteins we used for our collaborative painting, identify and explain at least one biophysical or functional property of each protein that affects expression or readout in cell-free systems. (Hint: options include maturation time, acid sensitivity, folding, oxygen dependence, etc) (1-2 sentences each)

Functional Properties of Fluorescent Proteins sfGFP (Superfolder GFP): This protein is engineered for extremely rapid folding and high stability, making it highly resistant to aggregation even when expressed at high rates in cell-free extracts.

mRFP1 (monomeric Red Fluorescent Protein): A key limitation is its relatively slow maturation time and lower photostability, which often results in a delayed fluorescence signal compared to green variants in short-term reactions.

mKO2 (monomeric Kusabira Orange 2): This protein features high pH sensitivity (acid sensitivity); its fluorescence can be significantly quenched if the cell-free reaction undergoes acidification due to metabolic byproduct accumulation.

mTurquoise2: Known for its exceptional quantum yield and brightness, it requires very precise folding conditions to achieve its maximum fluorescence intensity in a cyan readout.

mScarlet-I: This is a high-performance red protein that is notably oxygen-dependent for the final step of chromophore maturation, meaning insufficient aeration in the reaction vessel can limit its readout.

Electra2: Designed for enhanced photostability and rapid folding, this protein provides a very fast readout, though it can be sensitive to the ionic strength of the buffer (specifically magnesium levels) during the initial translation phase.

  1. Create a hypothesis for how adjusting one or more reagents in the cell-free mastermix could improve a specific biophysical or functional property you identified above, in order to maximize fluorescence over a 36-hour incubation. Clearly state the protein, the reagent(s), and the expected effect.

Protein: mKO2 (monomeric Kusabira Orange 2)

Reagents to Adjust: HEPES-KOH (Buffer) and Potassium Phosphate.

Hypothesis: Increasing the concentration of HEPES-KOH and Potassium Phosphate in the master mix will improve the fluorescence of mKO2 by enhancing the system’s buffering capacity.

Expected Effect: In a long 36-hour incubation, the cell-free reaction typically produces organic acids as byproducts of glucose and ribose metabolism. Since mKO2 is acid-sensitive, a drop in pH would normally quench its signal mid-incubation. By strengthening the buffer, we can maintain the pH near 7.5 for the entire duration, preventing the quenching of the chromophore and maximizing the cumulative fluorescence readout.