Week 11 HW: Bioproduction & Cloud Labs

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

At this point, I’m not entirely sure what I’ve contributed to, as the artwork changed quite a lot. I’ve added a few yellow and green pixels when we first got access to the board, but as time went it changed so much that I don’t think any of them were left in the same spots. I did like the collaborative aspect of it, but going forward, I kind of wish everyone could contribute to just one/two pixels that couldn’t be overwritten. That would make the pointing-out aspect of it so much easier

arrwork contribution arrwork contribution

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

E. coli Lysate Provides the cellular machinery necessary for both transcription and translation, including ribosomes, tRNAs, translation factors, and other essential proteins.

Salts/Buffer

Potassium Glutamate Acts as the primary osmolyte to maintain ionic strength and pH stability in the reaction mixture.

HEPES-KOH pH 7.5 Maintains optimal pH for enzyme activity and protein synthesis throughout the reaction.

Magnesium Glutamate Provides essential Mg²⁺ cofactor required for ribosome function, translation factors, and RNA stability.

Potassium phosphate monobasic & dibasic Work together as a phosphate buffer system to maintain pH stability and provide inorganic phosphate necessary for nucleotide synthesis.

Energy/Nucleotide System

Ribose: A pentose sugar that serves as the sugar backbone for nucleotide synthesis.

Glucose: Provides metabolic energy and carbon source; can be phosphorylated to enter glycolysis and the pentose phosphate pathway for energy generation.

AMP, CMP, GMP, UMP Nucleoside monophosphates that serve as building blocks for RNA synthesis during transcription and are recycled during the reaction.

Guanine A purine base that can be salvaged and converted into GMP, providing an alternative route for guanine nucleotide synthesis when needed.

Translation Mix (Amino Acids)

17 Amino Acid Mix Provides the standard amino acids (minus Tyr and Cys, which are listed separately) needed for protein synthesis during translation.

Tyrosine & Cysteine Included separately, likely because they require special handling (Tyr for fluorescent protein variants, Cys to prevent oxidation or for selenomethionine incorporation).

Additives

Nicotinamide: Acts as a precursor for NAD⁺/NADH, essential cofactors for redox reactions and energy metabolism within the cell-free system.

Backfill

Nuclease Free Water: Dissolves all components and serves as the reaction medium; nuclease-free formulation prevents degradation of RNA transcripts.

Main Differences:

1-hour PEP-NTP vs. 20-hour NMP-Ribose-Glucose Master Mixes The 1-hour optimized PEP-NTP master mix uses phosphoenolpyruvate (PEP) and nucleoside triphosphates (NTPs), which provide rapid, readily-available energy and nucleotides for quick transcription/translation reactions with high yields but shorter reaction windows. In contrast, the 20-hour NMP-Ribose-Glucose master mix uses nucleoside monophosphates (NMPs) along with glucose and ribose, creating a more sustainable, self-regenerating system where nucleotides and energy are generated on-demand through metabolic pathways, allowing the reaction to continue for much longer periods but requiring longer reaction times to reach equivalent protein yields. The longer format is designed for extended protein synthesis reactions, while the PEP-NTP system is optimized for speed and high initial productivity.

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

Part 1: Biophysical/Functional Properties of Each Fluorescent Protein

  1. sfGFP can fold in under 10 minutes, making it one of the fastest-maturing green fluorescent proteins.

  2. mRFP1 is reported to be a somewhat slowly-maturing monomer with low acid sensitivity.

  3. mKO2 is an orange fluorescent protein with moderate acid sensitivity

  4. In mTurquoise2, the reaction with oxygen is a rate-determining step (although folding contributes as well), and oxygen is necessary for generating the planar alkene of the fluorophore. In cell-free systems, limited oxygen availability—especially as the reaction progresses and consumes dissolved oxygen—can slow or halt chromophore maturation, limiting cyan fluorescence development

  5. mScarlet is a red fluorescent protein with moderate acid sensitivity, a fluorescence lifetime of 3.9 ns (the highest value recorded to date for mRFPs), and a quantum yield of 0.70; much higher than the quantum yield of other monomeric RFPs, resulting in the highest calculated brightness in the mRFP spectral class.

  6. Electra2 is a flavin-based fluorescent protein that functions independently from molecular oxygen—its fluorescence intensity is independent from molecular oxygen and remains stable under hypoxic conditions.

Part 2: Hypothesis for Optimization

Hypothesis: Optimizing mTurquoise2 Expression via Enhanced Oxygenation Protein of Focus: mTurquoise2 Reagent(s) to Adjust:

Add: Oxygen-rich buffer or increase aeration (inject air/oxygen into the reaction headspace or use a gas-permeable reaction chamber) Add: Catalase enzyme supplement (to manage hydrogen peroxide accumulation, which competes with oxygen utilization) Increase: Magnesium Glutamate concentration (higher Mg²⁺ can stabilize protein folding and chromophore maturation steps, especially when combined with oxygenation)

Expected Effect: Since the reaction with oxygen is a rate-determining step in mTurquoise2 maturation, increasing dissolved oxygen availability throughout the 36-hour incubation would:

Accelerate the rate-limiting oxidation step of chromophore maturation Generate peak cyan fluorescence earlier in the reaction timeline Maintain sustained fluorescence output even as reaction conditions evolve over 36 hours Prevent the typical oxygen-depletion bottleneck that limits cyan FP maturation in extended cell-free reactions

This approach leverages the known oxygen-dependency of mTurquoise2 maturation to directly address its biophysical limiting factor in a 36-hour cell-free context.

Alternative Hypothesis: Optimizing Electra2 for Cell-Free Viability via Flavin Supplementation Protein of Focus: Electra2 Reagent(s) to Adjust:

Add: Flavin Mononucleotide (FMN) or Flavin Adenine Dinucleotide (FAD) supplement (10–50 μM) Increase: Riboflavin concentration in the energy/nucleotide system (to replenish flavin pool as it’s consumed)

Expected Effect: Since Electra2 is a flavin-binding fluorescent protein, flavin mononucleotide (FMN) is essential to cell physiology as well as for determining fluorescence in flavin-based reporters. Supplementing exogenous FMN or FAD would:

Ensure adequate flavin cofactor availability throughout the 36-hour reaction Prevent signal loss due to flavin depletion (which occurs as the cell-free lysate consumes flavins for other metabolic processes) Maintain consistent blue fluorescence output independent of oxygen fluctuations Potentially enable higher protein expression levels by not competing for limited intracellular flavin pools

This approach addresses Electra2’s cofactor-dependent nature and leverages its oxygen-independence to provide a robust fluorescence readout over extended cell-free reactions.