Week 11 HW: Bioproduction & Cloud Labs

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

I missed the opportunity to contribute to the HTGAA CFPS bioart project. Later, I contributed to the SynBioBeta bioart project. I worked on part of the DNA on the center-left plate.

What I liked: This kind of community-coordinated experiment builds genuine shared investment in the outcome, which is a rare and valuable pedagogical achievement.
What could be improved: For future years, giving participants a low-resolution preview of the emerging canvas in near-real-time — without revealing the final image — would heighten the sense of collective emergence and encourage more strategic pixel placement.

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

1. Referencing the cell-free protein synthesis reaction composition E. coli Lysate

BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): The crude cell lysate supplies the entire transcription-translation (TX-TL) machinery — ribosomes, tRNA synthetases, elongation and initiation factors, chaperones, and co-factors — needed to convert DNA template into protein in vitro; the BL21 DE3 strain is specifically engineered to co-express T7 RNA Polymerase (from the chromosomal DE3 insertion), which is essential for driving transcription from the T7φ10 promoter used on most CFPS expression plasmids.

Salts/Buffer

Potassium Glutamate: The primary intracellular-mimicking monovalent cation that stabilises ribosome conformation and partially replaces KCl to avoid chloride-induced inhibition; potassium ions are critical for maintaining ribosome association and translational fidelity.

HEPES-KOH pH 7.5: A zwitterionic biological buffer that maintains reaction pH close to physiological values throughout the synthesis reaction, preventing the accumulation of protons that would otherwise inactivate ribosomes and enzymes as phosphate is metabolised.

Magnesium Glutamate: Provides free Mg²⁺ ions, which are indispensable cofactors for ribosome assembly, RNA polymerase activity, and the enzymatic reactions of the energy regeneration system; Mg²⁺ concentration is one of the most sensitive optimisation parameters in CFPS and must be carefully titrated for each lysate batch.

Potassium Phosphate Monobasic / Dibasic: Phosphate ions participate in energy metabolism and buffer secondary pH fluctuations; they also serve as inorganic phosphate donors and are required for metabolic pathways within the lysate.

Energy / Nucleotide System

Ribose: A five-carbon sugar that serves as the backbone of nucleoside synthesis; in the NMP-Ribose-Glucose system, ribose provides the sugar moiety for regenerating nucleoside monophosphates through the pentose phosphate pathway and purine/pyrimidine salvage enzymes present in the lysate.

Glucose: The primary carbon and energy source for ATP regeneration via glycolysis; endogenous glycolytic enzymes in the lysate convert glucose to pyruvate, generating ATP and NADH needed to sustain transcription and translation over extended reaction times.

AMP, CMP, GMP, UMP: Nucleoside monophosphates (NMPs) that serve as precursors for NTP synthesis; rather than supplementing expensive pre-formed NTPs, the NMP-based system relies on endogenous nucleoside monophosphate kinases (NMPK) and pyruvate kinase within the lysate to phosphorylate these precursors to the triphosphate form needed for transcription.

Guanine: A free purine base that feeds the guanosine salvage pathway; nucleoside phosphorylase enzymes in the lysate convert guanine + ribose-1-phosphate to guanosine, which is then phosphorylated to GMP and ultimately GTP — providing an additional low-cost input for GTP pools without supplying GMP directly.

Translation Mix (Amino Acids)

17 Amino Acid Mix: Provides the bulk of the 20 canonical amino acids needed for polypeptide elongation; splitting the amino acid supply allows independent supplementation of the three most problematic residues that tend to degrade, oxidise, or precipitate under standard CFPS conditions.

Tyrosine: Supplied separately because tyrosine has very low aqueous solubility at neutral pH and precipitates out of a premixed solution; it is added as a separately prepared suspension or at low concentration to ensure it remains bioavailable during the reaction.

Cysteine: Added separately because cysteine is prone to oxidative degradation — it dimerises to cystine under aerobic conditions — so it is freshly prepared and added just before the reaction to maintain a sufficient pool for proteins requiring cysteine residues.

Additives

Nicotinamide: A precursor to NAD⁺, which is an essential redox cofactor for glycolysis (particularly the GAPDH reaction); supplementing nicotinamide ensures the lysate can regenerate NAD⁺ from NADH during glucose catabolism, sustaining the energy regeneration capacity of the reaction over long incubations.

Backfill

Nuclease Free Water: Added to bring the reaction to the desired final volume without introducing RNases or DNases that would degrade the RNA polymerase transcript or the DNA template; using nuclease-free water is essential to protect the mRNA intermediates produced during transcription.

2. Describe the main differences between the 1-hour optimized PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix The 1-hour PEP–NTP system uses phosphoenolpyruvate (PEP) as a high-energy phosphate donor to rapidly regenerate ATP via pyruvate kinase and relies on pre-formed NTPs, producing a short, intense burst of energy that supports fast, high-yield protein synthesis before quickly terminating as PEP is depleted. In contrast, the 20-hour NMP–Ribose–Glucose system uses glucose-driven metabolism for continuous ATP regeneration and enzymatic phosphorylation of NMPs into NTPs, enabling slower but long-duration protein expression at lower cost and is preferred for sustained experiments.

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.

i. sfGFP (superfolder GFP)
sfGFP is engineered for very fast, robust β‑barrel folding, giving near-complete folding efficiency even when fused to aggregation-prone partners and making it a reliable baseline reporter for CFPS optimization. Its chromophore maturation requires molecular oxygen, so oxygen availability will affect fluorescence in cell-free reactions.

ii. mRFP1
mRFP1 has relatively slow chromophore maturation, meaning early-time fluorescence can substantially underreport total protein synthesis if fluorescence alone is used as a proxy. Its chromophore formation depends on molecular oxygen.

iii. mKO2
mKO2 is a fast-folding orange FP but retains acid sensitivity, so its fluorescence can be quenched if the cell-free reaction drifts acidic during prolonged incubations. This pH vulnerability should be monitored in long or metabolically active CFPS systems.

iv. mTurquoise2
mTurquoise2 is an intrinsically bright cyan FP with very high quantum yield, but native cysteines can form disulfide-linked oligomers in oxidising conditions, reducing the fraction of fluorescent protein. In cell-free systems, including reducing agents may be necessary, and its slower maturation versus sfGFP affects early readouts.

v. mScarlet-I
mScarlet-I is a bright, monomeric red FP with fast maturation and good photostability, making it a robust red reporter for long cell-free incubations and oxidising conditions. Its high intrinsic brightness supports sensitive fluorometric detection.

vi. Electra2
Electra2 is a relatively bright blue FP but blue FPs typically produce lower absolute signal than green or red FPs under equivalent expression, so overall fluorescence will be lower. It has reported tendencies to aggregate in some contexts, which can reduce soluble, fluorescent yield in CFPS.

2. 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 targeted: mKO2

Property identified: Acid sensitivity — mKO2 fluorescence is quenched at lower pH, and the NMP-Ribose-Glucose system can accumulate acidic byproducts over a 36‑hour incubation that may shift reaction pH downward.

Hypothesis: Increasing buffer capacity (HEPES-KOH from ~50 mM to ~100–150 mM and/or adding elevated potassium phosphate dibasic) will maintain pH ~7.2–7.8 and preserve mKO2 chromophore protonation; alternatively, reducing glucose to ~50% could slow acid production at the cost of some ATP regeneration.

Expected effect: Higher buffer capacity and/or reduced glucose should yield increased mKO2 fluorescence at 20–36 h versus standard mix, with a possible reduction in early (0–6 h) peak expression; test by plotting fluorescence kinetics across buffer and glucose gradients.