Week 11 HW: Bioproduction & Cloud Labs

Part A

Unfortunately, I did not have the opportunity to contribute to the project before the deadline ended. However, for next semester, I think it would be a good idea to create several variations of the same artwork using different color palettes or design concepts. I noticed that many people were unsure about what exact pattern or style they were supposed to contribute, while others had their own creative ideas that did not fully match the overall design. Because everyone has different artistic preferences and interpretations, it could be helpful to divide the project into multiple themed sections or versions. This would make the collaboration process more flexible, reduce confusion, and allow more students to express their creativity in their own way.

Cell-Free Reaction Component Roles (20-Hour System)

E. coli Lysate / BL21 (DE3) Star Lysate: Provides the essential molecular machinery, such as ribosomes and translation factors, required to synthesize proteins from an RNA template. The inclusion of T7 RNA Polymerase specifically enables high-level, targeted transcription of genes cloned under a T7 promoter.
Potassium Glutamate: Serves as the primary source of potassium ions to maintain correct intracellular ionic strength and supports optimal ribosome stability during translation. The glutamate anion acts as a compatible solute that mimics the physiological conditions of living bacterial cells.
HEPES-KOH pH 7.5: Functions as a chemical buffer to maintain a stable, optimal pH environment for enzymatic activity throughout the course of the prolonged incubation. It resists pH fluctuations that can occur as metabolic byproducts accumulate in the reaction.
Magnesium Glutamate: Supplies essential magnesium ions (Mg2+) which act as mandatory cofactors for the structural stability of ribosomes and the proper catalytic function of polymerases. Precise concentration management is critical, as magnesium directly influences translation accuracy and efficiency.
Potassium phosphate monobasic / dibasic: Forms a secondary buffering system that stabilizes pH while simultaneously providing inorganic phosphate ions (Pi). This phosphate source is crucial for driving the enzymatic recycling and phosphorylation of nucleotides into energy-rich forms.
Ribose: Functions as a stable carbohydrate precursor that is enzymatically processed within the reaction to synthesize the sugar backbones of nucleosides. This enables the sustainable, long-term generation of nucleotides over extended incubation periods.
Glucose: Serves as a primary metabolic energy source that undergoes catabolism to generate adenosine triphosphate (ATP) through glycolysis-like pathways. This continuous energy generation sustains the metabolic demands of transcription and translation over many hours.
AMP / CMP / GMP / UMP: Represent the nucleoside monophosphates (NMPs) that serve as basic building blocks for the reaction. They are dynamically phosphorylated into nucleoside triphosphates (NTPs) to power both transcription and ongoing energy-consuming translation steps.
Guanine: Acts as a purine base precursor that can be salvaged by the bacterial enzymes in the lysate to supplement the nucleotide pool. This ensures that guanosine-based energy intermediates (GTP) remain sufficient for the protein synthesis elongation steps.
17 Amino Acid Mix: Supplies the fundamental monomeric building blocks necessary for assembling the primary peptide chains during protein translation. This core mix lacks certain low-solubility or sensitive amino acids that must be prepared and adjusted independently.
Tyrosine: An aromatic amino acid added separately due to its poor solubility at neutral pH, which requires precise preparation (often at pH 12) to ensure adequate concentration in the final master mix. It is essential for incorporating tyrosine residues into the nascent protein chain.
Cysteine: A sulfur-containing amino acid added independently because it is highly prone to oxidation and degradation when stored in complex mixtures. It is vital for the formation of disulfide bonds and maintaining proper tertiary protein structures.
Nicotinamide: Acts as a stabilizing additive and precursor for pyridine nucleotides like NAD+, supporting the active metabolic pathways within the lysate. It helps maintain the redox balance required for sustained, long-term enzymatic energy regeneration.
Nuclease Free Water: Used to backfill the reaction to its final volume, ensuring that all chemical reagents are precisely diluted to their intended target concentrations. It is strictly purified to remove any degrading nucleases that could destroy the DNA template or RNA transcripts.

Key Differences: 1-Hour vs. 20-Hour Master Mixes

The 1-hour optimized master mix relies on pre-supplied nucleoside triphosphates (NTPs) and phosphoenolpyruvate (PEP) to deliver immediate, high-burst energy for rapid transcription and translation. Conversely, the 20-hour system utilizes low-cost, stable precursors—specifically nucleoside monophosphates (NMPs), ribose, and glucose—which are gradually converted into functional energy molecules by the lysate’s internal metabolic enzymes. This structural shift prevents early energy exhaustion and byproduct inhibition, allowing for a highly sustainable, cost-effective, and prolonged protein production window.

Cell-Free Reaction Component Roles (20-Hour System)

E. coli Lysate / BL21 (DE3) Star Lysate: Provides the essential molecular machinery, such as ribosomes and translation factors, required to synthesize proteins from an RNA template. The inclusion of T7 RNA Polymerase specifically enables high-level, targeted transcription of genes cloned under a T7 promoter.
Potassium Glutamate: Serves as the primary source of potassium ions to maintain correct intracellular ionic strength and supports optimal ribosome stability during translation. The glutamate anion acts as a compatible solute that mimics the physiological conditions of living bacterial cells.
HEPES-KOH pH 7.5: Functions as a chemical buffer to maintain a stable, optimal pH environment for enzymatic activity throughout the course of the prolonged incubation. It resists pH fluctuations that can occur as metabolic byproducts accumulate in the reaction.
Magnesium Glutamate: Supplies essential magnesium ions (Mg2+) which act as mandatory cofactors for the structural stability of ribosomes and the proper catalytic function of polymerases. Precise concentration management is critical, as magnesium directly influences translation accuracy and efficiency.
Potassium phosphate monobasic / dibasic: Forms a secondary buffering system that stabilizes pH while simultaneously providing inorganic phosphate ions (Pi). This phosphate source is crucial for driving the enzymatic recycling and phosphorylation of nucleotides into energy-rich forms.
Ribose: Functions as a stable carbohydrate precursor that is enzymatically processed within the reaction to synthesize the sugar backbones of nucleosides. This enables the sustainable, long-term generation of nucleotides over extended incubation periods.
Glucose: Serves as a primary metabolic energy source that undergoes catabolism to generate adenosine triphosphate (ATP) through glycolysis-like pathways. This continuous energy generation sustains the metabolic demands of transcription and translation over many hours.
AMP / CMP / GMP / UMP: Represent the nucleoside monophosphates (NMPs) that serve as basic building blocks for the reaction. They are dynamically phosphorylated into nucleoside triphosphates (NTPs) to power both transcription and ongoing energy-consuming translation steps.
Guanine: Acts as a purine base precursor that can be salvaged by the bacterial enzymes in the lysate to supplement the nucleotide pool. This ensures that guanosine-based energy intermediates (GTP) remain sufficient for the protein synthesis elongation steps.
17 Amino Acid Mix: Supplies the fundamental monomeric building blocks necessary for assembling the primary peptide chains during protein translation. This core mix lacks certain low-solubility or sensitive amino acids that must be prepared and adjusted independently.
Tyrosine: An aromatic amino acid added separately due to its poor solubility at neutral pH, which requires precise preparation (often at pH 12) to ensure adequate concentration in the final master mix. It is essential for incorporating tyrosine residues into the nascent protein chain.
Cysteine: A sulfur-containing amino acid added independently because it is highly prone to oxidation and degradation when stored in complex mixtures. It is vital for the formation of disulfide bonds and maintaining proper tertiary protein structures.
Nicotinamide: Acts as a stabilizing additive and precursor for pyridine nucleotides like NAD+, supporting the active metabolic pathways within the lysate. It helps maintain the redox balance required for sustained, long-term enzymatic energy regeneration.
Nuclease Free Water: Used to backfill the reaction to its final volume, ensuring that all chemical reagents are precisely diluted to their intended target concentrations. It is strictly purified to remove any degrading nucleases that could destroy the DNA template or RNA transcripts.

Key Differences: 1-Hour vs. 20-Hour Master Mixes

Part C

Fluorescent Protein Properties in Cell-Free Systems

sfGFP (Superfolder GFP): sfGFP is engineered to fold very efficiently, even when the environment is a bit stressful or crowded. This means most of the protein that is translated in a cell‑free lysate becomes properly folded and fluorescent very quickly, so signal builds up fast.
mRFP1: mRFP1 has slower and less efficient maturation than newer red FPs, so freshly made protein often sits in non‑ or weakly fluorescent intermediate states for a while. As a result, early time‑points in a cell‑free experiment can show relatively low red signal even when translation itself is strong
mKO2: mKO2 depends on oxygen to complete its chromophore maturation, so its fluorescence strongly reflects how much O₂ is available during the reaction. In sealed or poorly mixed plates, oxygen can become limiting over time, and the orange signal will level off or stay low even if more protein continues to be produced.
mTurquoise2: mTurquoise2 is a very bright cyan protein with high quantum yield, so each correctly folded molecule gives a strong signal. However, its folding is somewhat demanding, so if the lysate has limited chaperone activity or suboptimal conditions, a noticeable fraction of the translated protein may misfold and never become fluorescent.
mScarlet_I: mScarlet_I combines high brightness with relatively fast maturation for a red FP, which makes it good for time‑course measurements in cell‑free systems. Its fluorescence, however, drops when the reaction becomes more acidic, so pH drift during long incubations can reduce the apparent signal even without changes in expression
Electra2: Electra2 matures very quickly, so blue fluorescence appears early and can track expression dynamics on short timescales. Over long experiments with repeated plate‑reader scans, its moderate photostability means the signal can fade due to photobleaching, making late‑time measurements underestimate the amount of protein present

Optimization Hypothesis for a 36-Hour Incubation

Proteins: mScarlet_I & mKO2 Reagents to Adjust: HEPES-KOH (Buffer) and oxygen

Hypothesis: Increasing the concentration of HEPES-KOH while supplementing the system with an active oxygenation will maximize the long-term fluorescence readout of mScarlet_I and mKO2 over a 36-hour incubation.

Expected Effect: A stronger buffer should reduce pH drops caused by metabolism in the lysate, and because mScarlet_I is sensitive to acidic conditions, this should help maintain a brighter and more consistent signal over time.