Week 11

Class Assignment — Week 11

Part A. Community Bioart Reflections | The 1,536 Pixel Artwork Canvas

I contributed to the “Love” apple-shaped yellow sign at the mid-bottom of the artwork, working on the DNA assembly for that section of the plate.

What I liked most is the premise itself: that biology can be a medium for public communication, not just a laboratory tool. There is something genuinely powerful about a piece of art that is also a functional scientific artefact — 1,536 colonies, four colours, four quadrants, one coherent image, built by 154 people across 7,946 individual contributions. Projects like this do more for science outreach than most formal presentations ever will, because they meet people where curiosity lives. The collaborative structure reinforced that too. No single person could have produced this at scale. Every contribution, however small, was load-bearing. That is a lesson worth carrying into research.

For next year, a few things could sharpen the experience. The process deserves better documentation — annotated diagrams of who contributed what quadrant and colour, and a short write-up of the biological design logic mapping colony colour to fluorescent protein or pigment pathway. That record becomes an outreach asset in its own right, and for participants from under-resourced contexts it also serves as tangible evidence of having done real science. I would also push for a clearer throughline between the artistic concept and the biology: why this sequence, why this organism, why this visual. That conceptual anchoring is what separates bioart that educates from bioart that merely looks interesting from a distance.

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

Cell-Free Reaction Components (20-Hour NMP-Ribose Master Mix)

E. coli Lysate

BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): The lysate is the reaction engine. It supplies the ribosomes, translation factors, chaperones, and metabolic enzymes needed to carry out transcription and protein synthesis. The DE3 strain harbours a chromosomal T7 RNA Polymerase gene, so the lysate comes pre-loaded with the polymerase needed to drive T7 promoter-based expression.

Salts/Buffer

Potassium Glutamate: The primary monovalent salt. It maintains ionic strength and stabilises ribosome conformation while also serving as a mild crowding agent that mimics the intracellular environment.

HEPES-KOH pH 7.5: The buffering system. It holds the reaction at a physiologically permissive pH, which matters because both ribosome activity and enzyme kinetics are sensitive to even modest pH drift over a 20-hour incubation.

Magnesium Glutamate: Magnesium is indispensable for ribosome assembly and catalytic activity. It also stabilises nucleotide triphosphates and is a cofactor for many of the enzymes active in the lysate.

Potassium Phosphate (monobasic and dibasic, 1.6:1 ratio): The phosphate pair serves dual duty: secondary pH buffering and phosphate donor pool. The specific dibasic:monobasic ratio fine-tunes the buffering capacity at pH 7.5 and feeds into nucleotide regeneration pathways.

Energy / Nucleotide System

Ribose: The carbon backbone for nucleotide biosynthesis. Cellular enzymes in the lysate phosphorylate and elaborate ribose into the nucleotide monophosphates needed for RNA synthesis, making it the upstream feedstock for the whole energy system.

Glucose: A supplementary carbon and energy source. It feeds into glycolysis within the lysate to regenerate ATP and sustain metabolic activity over the extended 20-hour window.

AMP, CMP, UMP: Nucleotide monophosphate precursors. The lysate enzymes phosphorylate these to their di- and triphosphate forms, supplying the NTPs required for transcription without the instability problems associated with adding NTPs directly.

GMP: Absent from this mix (0.00 uM in the image). Guanine is supplied instead and salvaged into GMP by the lysate’s purine salvage pathway, making direct GMP supplementation unnecessary.

Guanine: The free base precursor for guanosine nucleotides. Lysate hypoxanthine-guanine phosphoribosyltransferase (HGPRT) converts it to GMP via the purine salvage pathway, which is then phosphorylated to GDP and GTP for use in transcription.

Translation Mix (Amino Acids)

17 Amino Acid Mix: The bulk substrate pool for translation. Seventeen of the twenty standard amino acids are supplied together; tyrosine and cysteine are handled separately because of their solubility and stability constraints.

Tyrosine: Supplied at elevated pH (pH 12 stock) because tyrosine has very low aqueous solubility at neutral pH. It is added separately to avoid precipitation in the master mix.

Cysteine: Also added separately due to its tendency to oxidise in bulk amino acid stocks, which would render it unusable for translation. Keeping it isolated until reaction assembly preserves its reduced form.

Additives

Nicotinamide: An NAD+ precursor and sirtuin inhibitor. It helps maintain the NAD+/NADH redox balance needed to sustain metabolic enzyme activity across the long incubation, and may also reduce non-specific protein degradation by inhibiting NAD+-dependent deacylases in the lysate.

Backfill

Nuclease-Free Water: Brings the reaction to final volume without introducing RNases that would degrade the mRNA template and collapse expression.

Question 1: Key Differences Between the 1-Hour PEP-NTP and 20-Hour NMP-Ribose Master Mixes

The 1-hour PEP-NTP system supplies energy and nucleotides directly: preformed NTPs (ATP, GTP, CTP, UTP) plus phosphoenolpyruvate (PEP-Mono) as the immediate phosphate donor for ATP regeneration, with maltodextrin as a secondary carbon source. This makes it fast but metabolically shallow since the NTP pool is fixed at the start and depletes without robust regeneration. The 20-hour NMP-Ribose system takes the opposite approach: it supplies nucleotide monophosphates and simple sugars (ribose, glucose) as upstream precursors, letting the lysate’s own enzymes synthesise and continuously regenerate NTPs throughout the reaction, which sustains expression over a far longer window. The additives also diverge sharply: the 1-hour mix includes spermidine, DMSO, cAMP, NAD, and folinic acid to boost immediate transcription/translation efficiency, while the 20-hour mix strips these down to nicotinamide alone, reflecting a design philosophy of metabolic sustainability over peak output.

Bonus: How Does Transcription Occur If GMP Is 0.00 uM?

GMP is listed at 0.00 uM because it is not supplied directly. Guanine is present instead, and the lysate’s purine salvage machinery, specifically HGPRT, converts free guanine to GMP using PRPP (phosphoribosyl pyrophosphate) as the ribose-phosphate donor. That GMP is then phosphorylated to GDP and GTP by nucleoside monophosphate kinases and pyruvate kinase respectively. The system effectively outsources GTP synthesis to the lysate’s own enzymes rather than paying the cost of supplying pre-formed GMP that could be unstable or inhibitory at high concentrations.

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

Fluorescent Protein Biophysical Properties (20-Hour NMP-Ribose Master Mix)

1. sfGFP

sfGFP was specifically engineered for robust folding under conditions where normal GFP would misfold or aggregate. It showed a 3.5-fold faster initial refolding rate than its parent frGFP and tolerated higher denaturant concentrations , which directly translates to better performance in the crowded, chaperone-limited environment of a cell-free lysate. In a 36-hour reaction, that folding robustness means a higher fraction of translated protein reaches a fluorescent state rather than being lost to misfolding.

2. mRFP1

The most relevant property here is incomplete chromophore maturation. mRFP1 shows two absorption peaks at 503 nm and 584 nm; the 503 nm peak corresponds to a green fraction that never fully matures beyond the green intermediate, with a quantum yield of only 0.27. In a cell-free system, there is no cellular quality control or folding assistance to rescue this incomplete maturation fraction, so a meaningful portion of expressed mRFP1 will likely remain dim or spectrally contaminated, reducing effective red fluorescence yield over the 36-hour incubation.

3. mKO2

mKO2 is a fast-folding variant of mKO1, engineered with 8 additional mutations for rapid maturation, though it has moderate acid sensitivity. The acid sensitivity is the property most relevant to cell-free. As the NMP-Ribose reaction runs over 36 hours, metabolic byproducts can acidify the reaction environment, and even modest pH drift below 7.0 could reduce mKO2 fluorescence output. Buffering capacity of the HEPES-KOH system is critical here specifically for mKO2.

4. mTurquoise2

mTurquoise2 has a maturation half-time of approximately 36.5 minutes , which is slow relative to other cyan variants. In a short reaction this would be a problem, but over 36 hours it is unlikely to be the bottleneck. The more relevant consideration is its complex, multi-step maturation kinetics: mTurquoise2 shows complex maturation kinetics requiring more than one kinetic step , meaning the protein accumulates through intermediate states before reaching peak fluorescence. For a 36-hour readout, this matters less than it would for a 1-hour endpoint assay.

5. mScarlet-I

mScarlet-I is one of the brightest monomeric red fluorescent proteins currently available, but it carries a known photostability limitation. The photostability of mScarlet-I is lower than mCherry under FRET imaging conditions, though under typical dynamic experiment conditions it barely loses intensity. More relevant to cell-free is that all GFP-like chromophores, including mScarlet-I’s, require molecular oxygen for maturation. In a sealed 20 uL reaction running for 36 hours, dissolved oxygen will be consumed early, meaning late-translated mScarlet-I molecules may not fully mature. This is probably the single biggest performance limiter for the red channel over long incubations.

6. Electra2

Electra2 is a blue fluorescent protein derived from mRuby3, engineered through hierarchical screening in bacterial and mammalian cells, with excitation at 403 nm and emission at 456 nm. Quantification of intracellular brightness showed Electra2 was approximately 2.1 times brighter than mTagBFP2 , which is impressive for the blue channel. The key biophysical caveat is that, like all GFP-derived beta-barrel FPs, Electra2 still requires molecular oxygen for chromophore maturation. This makes oxygen depletion over 36 hours a shared limitation with mScarlet-I, and potentially more acute for Electra2 because blue-channel chromophore formation is generally less efficient than green or red.

Hypothesis: Improving mScarlet-I Fluorescence Over 36-Hour Incubation

Protein: mScarlet-I

Problem: Oxygen-dependent chromophore maturation means late-translated mScarlet-I molecules cannot mature in a sealed, metabolically active reaction where dissolved O2 is consumed within the first few hours.

Hypothesis: Supplementing the 2 uL custom reagent slot with a controlled headspace oxygen carrier, specifically a dilute catalase-free perfluorocarbon oxygen supplement or simply increasing the dissolved O2 pre-reaction by briefly aerating the master mix before sealing, would extend the oxygen availability window and increase the proportion of mScarlet-I that reaches full chromophore maturation. Practically, within the reaction composition (6 uL lysate + 10 uL master mix + 2 uL DNA + 2 uL supplements), the 2 uL supplement volume could carry a small amount of hydrogen peroxide at sub-millimolar concentration as a slow O2 donor, with catalase from the lysate itself releasing O2 gradually throughout the incubation. Expected effect: higher peak fluorescence and a later-onset fluorescence plateau, reflecting maturation of protein translated in the middle and later phases of the 36-hour window rather than only the early burst.