Week 11 — Bioproduction & Cloud Labs

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

  1. Done :) global artwork experiment
  2. Make a note on your HTGAA webpages including:
    • I was part of the community bioart project by supporting others design.
    • I like collaborative artworks because of their inclusive atmosphere.
    • I don’t have any recommendations.

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

1. Roles of Each Cell-Free Reaction Component

E. coli Lysate BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) The lysate provides the molecular machinery required for transcription and translation, including ribosomes, tRNAs, metabolic enzymes, and cofactors. T7 RNA polymerase specifically drives strong transcription from T7 promoters, enabling efficient protein production.

Salts / Buffer Potassium Glutamate Potassium glutamate maintains ionic strength and mimics the intracellular environment of E. coli. It stabilizes ribosome activity and improves translation efficiency.

HEPES-KOH pH 7.5 HEPES acts as a buffering agent that maintains a stable pH during the reaction. Stable pH is essential because enzyme activity and protein folding are highly pH-sensitive.

Magnesium Glutamate Magnesium ions are essential cofactors for ribosomes, RNA polymerases, and ATP-utilizing enzymes. Proper Mg²⁺ concentration strongly affects transcription, translation, and protein folding.

Potassium Phosphate Monobasic / Dibasic These phosphate salts contribute to buffering capacity and phosphate balance. Together they help stabilize reaction conditions during long incubations.

Energy / Nucleotide System Ribose Ribose supports nucleotide regeneration and energy metabolism pathways. It helps sustain longer protein synthesis reactions.

Glucose Glucose serves as an energy substrate that can regenerate ATP through glycolytic enzymes remaining in the lysate. This extends reaction longevity.

AMP, CMP, GMP, UMP These nucleotides are the building blocks for RNA synthesis during transcription. RNA polymerase incorporates them into mRNA transcripts.

Guanine Guanine can be salvaged enzymatically into GMP inside the lysate. This reduces reagent cost while still supporting transcription.

Translation Mix (Amino Acids) 17 Amino Acid Mix This mixture supplies most amino acids required for protein synthesis. Ribosomes use them to elongate nascent polypeptide chains.

Tyrosine Tyrosine is supplied separately because it can degrade or precipitate under storage conditions. Maintaining correct tyrosine levels improves protein yield.

Cysteine Cysteine is added separately because it is chemically unstable and easily oxidized. It is critical for proper disulfide bond formation and protein folding.

Additives Nicotinamide Nicotinamide supports redox and metabolic enzyme activity by contributing to NAD-related biochemical pathways. This can improve reaction stability and protein yield over longer incubations.

Backfill Nuclease-Free Water Nuclease-free water adjusts the final reaction volume while preventing RNA or DNA degradation by contaminating nucleases.

2. Difference Between the 1-Hour PEP-NTP Mix and 20-Hour NMP-Ribose-Glucose Mix

The 1-hour optimized PEP-NTP system is designed for rapid, high-yield protein expression using phosphoenolpyruvate (PEP) as a fast ATP regeneration source and direct nucleotide triphosphates (NTPs) for transcription. In contrast, the 20-hour NMP-ribose-glucose system uses lower-cost nucleotide monophosphates and slower metabolic energy regeneration pathways, enabling longer and more economical protein synthesis reactions.

The long-duration mix sacrifices some initial expression speed in exchange for improved sustainability and reduced reagent cost, making it more suitable for large-scale or extended incubations.

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

Biophysical or Functional Properties Affecting Cell-Free Expression

  1. sfGFP

sfGFP matures rapidly and folds extremely efficiently, even under partially stressful conditions. Its robust folding makes it highly compatible with cell-free systems and long incubations.

  1. mRFP1

mRFP1 has a relatively slower chromophore maturation time compared to GFP derivatives. This delayed maturation can reduce apparent fluorescence during short incubation experiments.

  1. mKO2

mKO2 is sensitive to acidic environments and requires oxygen-dependent chromophore maturation. Changes in pH or oxygen availability may significantly affect fluorescence intensity.

  1. mTurquoise2

mTurquoise2 has extremely high quantum yield and brightness but requires efficient folding for optimal fluorescence. Magnesium concentration and incubation temperature may influence its folding efficiency.

  1. mScarlet_I

mScarlet_I is highly bright with improved maturation kinetics compared to older red fluorescent proteins. However, red chromophores generally mature more slowly than green fluorescent proteins.

  1. Electra2

Electra2 likely exhibits specialized spectral properties but may have increased sensitivity to oxidation or folding stress. Extended incubations could improve maturation if energy supply remains stable.

The amino acid sequences are shown in the HTGAA Cell-Free Benchling folder.

Hypothesis for Master Mix Optimization

  • Hypothesis

Increasing magnesium glutamate concentration and supplementing additional glucose in reactions expressing mScarlet_I will improve fluorescence intensity over a 36-hour incubation.

  • Reasoning

Higher magnesium levels may enhance ribosome stability and improve translation efficiency, while additional glucose can sustain ATP regeneration during long incubations. Because mScarlet_I has slower chromophore maturation kinetics, prolonged energy availability should increase total mature fluorophore formation and therefore increase fluorescence output.

  • Expected Effect

The modified master mix is expected to produce: -higher total fluorescence intensity, -improved protein yield, -and increased mature chromophore accumulation after 36 hours.

Example Custom Reagent Supplement Strategy

For long-term expression of mTurquoise2:

Add supplemental magnesium glutamate (+2–4 mM) Add additional glucose Include mild molecular crowding agents such as PEG-8000

This combination may improve folding efficiency, sustain ATP production, and stabilize fluorescence during extended incubation.

Data Analysis Strategy

After fluorescence measurements are collected, the data can be analyzed by comparing fluorescence intensity across different reagent formulations and incubation times. Statistical analysis can identify which reagent compositions maximize protein brightness, maturation efficiency, and long-term stability in cell-free systems.

Fluorescence curves over time can also reveal whether specific proteins benefit more from enhanced energy systems, altered magnesium concentrations, or improved folding environments.

Total: 20 μL reaction