Week 11 HW: Building Genomes

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

  1. Contribute at least one pixel to this global artwork experiment before the editing ends on Sunday 4/19 at 11:59 PM EST.
    • A personalized URL was sent to the email address associated with your Discourse account, and you can discuss the artwork on the Discourse.
    • If you did not have a chance to contribute, it’s okay, just make sure you become a TA this fall! 😉
  2. Make a note on your HTGAA webpages including:
    • what you contributed to the community bioart project (e.g., “I made part of the DNA on the bottom right plate”)
    • what you liked about the project, and
    • what about this collaborative art experiment could be made better for next year.'

I have participated in HTGAA Bioart 2026. It’s really amazing. Seems like, Science is not ONLY series; but also FUN. image.png image.png

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

image.png image.png 1. Referencing the cell-free protein synthesis reaction composition (the middle box outlined in yellow on the image above, also listed below), provide a 1-2 sentence description of what each component’s role is in the cell-free reaction.

E. coli Lysate

  • BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) provides the entire cellular machinery for transcription and translation, including ribosomes, tRNAs, aminoacyl-tRNA synthetases, elongation factors, and native metabolic enzymes. The “Star” strain carries an rne131 mutation that reduces mRNA degradation, and the integrated T7 RNA polymerase drives high-level transcription from T7 promoter templates.

Salts/Buffer

  • Potassium Glutamate supplies potassium ions essential for ribosome assembly, tRNA folding, and enzyme activity, while glutamate acts as a compatible counterion that stabilizes proteins better than chloride.

  • HEPES-KOH pH 7.5 a zwitterionic buffer that maintains the reaction near physiological pH, preventing acidification as metabolism generates protons and organic acids.

  • Magnesium Glutamate delivers Mg²⁺, a critical cofactor for ribosome structural integrity, tRNA–mRNA codon pairing, polymerase activity, and virtually all phosphoryl transfer reactions.

  • Potassium phosphate monobasic and Potassium phosphate dibasictogether they form a secondary phosphate buffer system and, more importantly, supply inorganic phosphate (Pᵢ) needed for substrate-level phosphorylation in central carbon metabolism to regenerate ATP from ADP.

Energy / Nucleotide System

  • Ribose - The primary carbon and energy source; it is phosphorylated and funneled through the pentose phosphate and glycolytic pathways by lysate enzymes to regenerate ATP and supply ribose-5-phosphate for nucleotide salvage

  • Glucose - A secondary energy substrate that feeds glycolysis to boost ATP regeneration and help sustain the long 20 hour incubation window.

  • AMP,CMP, UMP - Nucleoside monophosphate precursors that are salvaged and phosphorylated in situ to ATP, CTP, and UTP, providing the NTP pools required for transcription and translation in a cost-effective, sustainable way.

  • GMP - Intentionally omitted because GTP is instead generated from free guanine via the salvage pathway, which proved more efficient in the Ginkgo and OpenAI optimization.

  • Guanine - The free base precursor for GMP and GTP biosynthesis through the purine salvage pathway, replacing direct GMP supplementation as the guanine nucleotide source.

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix provides the canonical amino acid building blocks (excluding tyrosine and cysteine, which are supplied separately due to solubility constraints) that are charged onto tRNAs for polypeptide synthesis.

  • Tyrosine added separately in a high pH stock because tyrosine is poorly soluble at neutral pH; it completes the aromatic amino acid pool needed for translation.

  • Cysteine supplied separately to prevent premature oxidation and disulfide formation in the amino acid stock, ensuring availability for proteins containing cysteine residues.

Additive

  • Nicotinamide- A precursor for NAD⁺ and NADP⁺ biosynthesis and salvage, maintaining redox cofactor pools needed for the central metabolic reactions that regenerate ATP during the extended incubation.

Backfill

  • Nuclease Free Water brings the reaction to its final volume while avoiding contaminating RNases or DNases that would degrade the mRNA template or DNA, preserving transcription and translation fidelity.

2. Describe the main differences between the 1-hour optimized PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix shown in the Google Slide above. (2-3 sentences) The 1-hour PEP/NTP mix delivers energy and nucleotides directly as pre-made ATP, GTP, CTP, UTP, and PEP-Mono (with maltodextrin, spermidine, DMSO, cAMP, NAD, and folinic acid as additives), producing a fast but short-lived burst of protein synthesis before PEP is exhausted and phosphate accumulation inhibits the reaction.

The 20-hour NMP-Ribose-Glucose mix instead supplies indirect precursors, ribose and glucose for energy and nucleoside monophosphates (AMP, CMP, UMP) plus free guanine for NTP regeneration, relying on the lysate’s native salvage and glycolytic enzymes to sustainably regenerate ATP and NTPs over a much longer window.

The practical trade-off is speed versus sustainability and cost: PEP/NTP is expensive and front-loaded for rapid yield, while the NMP-Ribose system is cheaper, runs on simpler precursors, and uses a phosphate-buffered system to support extended incubation ideal for artwork-scale fluorescent protein production.

3. Bonus question: How can transcription occur if GMP is not included but Guanine is? Transcription can still proceed because E. coli lysate contains an intact purine salvage pathway that converts free guanine into GTP in situ. Specifically, the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT, encoded by gpt or hpt) attaches guanine to phosphoribosyl pyrophosphate (PRPP, derived from the ribose supplied in the mix) to form GMP directly. That GMP is then sequentially phosphorylated by guanylate kinase (Gmk) to GDP using ATP, and nucleoside diphosphate kinase (Ndk) to GTP, again using ATP as the phosphate donor. Since ATP is being continuously regenerated from AMP, ribose, and glucose through glycolysis and the pentose phosphate pathway, the lysate effectively runs its own small GTP factory, supplying T7 RNA polymerase with the GTP it needs to initiate and elongate mRNA transcripts. This salvage route is more cost effective than buying purified GMP outright and, as the Ginkgo and OpenAI optimization showed, actually outperforms direct GMP supplementation in sustained reactions.

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. (Hint: options include maturation time, acid sensitivity, folding, oxygen dependence, etc) (1-2 sentences each)

  1. sfGFP - Rapid folding and robust maturation. sfGFP was engineered with stabilizing mutations (S30R, Y39N, N105T, Y145F, I171V, A206V) that give it an exceptionally fast folding rate and a maturation half-time of roughly 6 minutes, making it ideal for short cell-free reactions where misfolded aggregates would otherwise dominate the readout. It also tolerates fusion to poorly folding partners, which is why it is the default benchmark protein for CFPS yield measurements.
  2. mRFP1 - Slow maturation and modest brightness. mRFP1 has a maturation half-time near 60 minutes and a relatively low quantum yield (~0.25) with extinction coefficient around 50,000 M⁻¹cm⁻¹, meaning fluorescence signal lags well behind protein synthesis and appears dim compared to newer red variants. In a 20 hour reaction this is acceptable, but in a 1 hour PEP reaction the chromophore barely finishes maturing before the energy system collapses.
  3. mKO2 - Acid sensitivity and oxygen dependent chromophore formation. mKO2 has a pKa of about 5.5, so it dims noticeably as the cell-free reaction acidifies from accumulating phosphate and organic acids during extended incubations, and like all GFP family derivatives its chromophore cyclization requires molecular O₂, so poorly aerated reaction vessels reduce signal. Its maturation is moderate (~trib 4500 minutes at 37 °C), which pairs well with the 20 hour NMP-Ribose format.
  4. mTurquoise2 - High quantum yield and pH stability. mTurquoise2 boasts the highest quantum yield (~0.93) of any cyan fluorescent protein, giving bright signal even at modest expression levels, and its pKa of ~3.1 makes it essentially immune to the pH drift that plagues cell-free reactions as metabolism progresses. Its monoexponential fluorescence lifetime also makes it the gold standard FRET donor if multiplexed readouts are desired.
  5. mScarlet_I - Fast maturation tuned for speed over brightness. mScarlet-I is the “improved kinetics” variant of mScarlet, trading some quantum yield (0.54 vs 0.70) for a dramatically shortened maturation half-time of roughly 36 minutes, which is critical because red chromophore formation requires an extra oxidation step beyond green FPs. This makes it the most practical red choice for cell-free painting where signal needs to appear within the reaction window rather than hours later.
  6. Electra2 - Bilirubin dependent, oxygen independent fluorescence. Electra2 is a UnaG derived fluorescent protein that binds the endogenous ligand bilirubin as its chromophore rather than autocatalytically forming one, which means it fluoresces without requiring molecular oxygen and matures essentially as fast as bilirubin can diffuse in. The catch for cell-free work is that bilirubin must be supplied exogenously to the reaction, since the E. coli lysate does not produce it, but once added the signal is nearly instantaneous and works in anaerobic or sealed reaction formats where GFP family proteins fail.

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

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 addressed: Acid sensitivity (pKa ~5.5), which causes progressive fluorescence loss as the cell-free reaction acidifies over long incubations due to phosphate accumulation and organic acid buildup from ribose and glucose catabolism.

Reagent adjustment: Increase HEPES-KOH pH 7.5 from 45 mM to approximately 100 mM, and simultaneously rebalance the potassium phosphate dibasic to monobasic ratio from the current symmetric 5.6 mM / 5.6 mM configuration to a dibasic heavy 8.0 mM / 3.2 mM split, raising the effective starting pH closer to 7.6 and increasing total buffering capacity near physiological pH.

Expected Effect By nearly doubling the HEPES buffering capacity and tilting the phosphate system toward its dibasic form, the reaction should resist the acidification that typically drops pH from ~7.5 to below 6.5 during 24 to 36 hour incubations. Because mKO2 fluorescence drops sharply below its pKa of 5.5, maintaining the bulk pH above 7.0 throughout the run should keep the chromophore predominantly in its bright protonation state, preserving quantum yield and extinction coefficient near their maxima for the entire 36 hour window rather than only the first 10 to 15 hours.

Predicted Outcome I would expect mKO2 fluorescence at the 36 hour endpoint to increase by roughly 2 to 3 fold compared to the unmodified 20 hour formulation scaled up, with the brightness curve plateauing rather than declining after hour 20. A secondary benefit is that mRFP1 and mScarlet-I, which also have mild acid sensitivity (pKa ~4.5 and ~5.4 respectively), should show modest signal preservation as well, giving the collaborative painting better color fidelity across the orange and red channels.

Control and Readout The cleanest test is a side by side kinetic read on a plate reader (ex 551 nm, em 565 nm for mKO2) comparing the standard 20 hour formulation against the buffer boosted 36 hour formulation, with a parallel pH time course using a microelectrode or pH sensitive dye like SNARF-1 to confirm the buffering hypothesis is actually the mechanism rather than some off target effect on ribosome activity or enzyme kinetics.

Trade-off to Watch Higher HEPES concentrations can slightly inhibit translation by altering ionic strength, so the potassium glutamate concentration may need to be trimmed by 20 to 30 mM to keep total osmolarity in the sweet spot for ribosome function. This is a classic cell-free optimization tension: you are trading a small hit to initial translation rate for a much larger gain in sustained fluorescence readout, which is exactly the right bet for a 36 hour artwork incubation where endpoint brightness matters far more than early kinetics.

3. The second phase of this lab will be to define the precise reagent concentrations for your cell-free experiment. You will be assigned artwork wells with specific fluorescent proteins and receive an email with instructions this week (by April 24). You can begin composing master mix compositions here. Here is a proposed 36 hour artwork master mix composition, written as a working draft you can adapt once your specific fluorescent protein assignments arrive. The design philosophy is simple: take the proven 20 hour NMP-Ribose formulation as the backbone, then tune the buffering, energy, and cofactor components to extend the productive window out to 36 hours while protecting pH sensitive chromophores like mKO2, mScarlet-I, and mRFP1.

For the Salts and Buffer block, I would set potassium glutamate at 290 mM (trimmed slightly from 312.6 mM to offset the osmotic load of extra HEPES), HEPES-KOH pH 7.5 at 100 mM (more than doubled from 45 mM for sustained pH control), magnesium glutamate at 7.5 mM (a touch above the 20 hour value to support the longer transcription window), and shift the phosphate system to potassium phosphate dibasic at 8.0 mM and monobasic at 3.2 mM, which biases starting pH toward 7.6 and gives the reaction more headroom before acidification drops it below the acid sensitive protein pKa values. The total phosphate pool stays near 11 mM, preserving its role as a substrate level phosphorylation substrate for ATP regeneration.

For the Energy and Nucleotide System, I would bump ribose to 90 mM (up from 77.4 mM) and glucose to 10 mM (up from 6.9 mM) to provide a larger carbon and phosphate donor reservoir for the extended run, keeping in mind that the lysate’s glycolytic and pentose phosphate enzymes are what convert these into ATP over time. The nucleoside monophosphates would stay close to the published optimum at AMP 600 µM, CMP 500 µM, UMP 500 µM (modest increases on CMP and UMP to feed the longer mRNA production window), GMP at 0 µM, and guanine raised to 300 µM to ensure the purine salvage pathway can keep GTP pools topped up for the full 36 hours. A small inorganic pyrophosphatase spike in (0.1 U/µL) is worth considering if available, because PPi accumulation from continuous NTP polymerization is a known late stage inhibitor.

For the Translation Mix, I would raise the 17 amino acid mix to 5.0 mM, tyrosine pH 12 to 5.0 mM, and cysteine to 5.0 mM, roughly a 20 percent boost over the 20 hour formulation to avoid amino acid depletion before the 36 hour mark. Amino acids are consumed stoichiometrically with protein synthesis, so a longer run simply needs a deeper pool, and the extra cysteine helps buffer against oxidation over the extended aerobic incubation.

For the Additives, I would keep nicotinamide at 3.10 mM to sustain NAD and NADP salvage, and add two targeted supplements: bilirubin at 50 µM (essential if any Electra2 wells are assigned, since it is an obligate ligand the lysate cannot synthesize), and putrescine or spermidine at 1 mM to stabilize ribosomes and mRNA structure during the long incubation, borrowing a trick from the 1 hour PEP formulation without adding the high energy PEP itself. A low dose of DTT at 1 mM is also worth considering to keep cysteine residues reduced over 36 hours of exposure to atmospheric oxygen.

The lysate itself should be BL21 (DE3) Star at roughly 30 percent final volume, consistent with typical CFPS protocols, and DNA template added at 5 to 10 nM for plasmid or 10 to 20 nM for linear PCR product, with the final volume brought up with nuclease free water as the backfill. Once the email arrives with the specific well assignments and fluorescent protein pairings, the main parameters to finalize are the template concentration per color (brighter proteins like sfGFP and mTurquoise2 can go lower, dimmer ones like mRFP1 should go higher), whether any wells need bilirubin for Electra2, and whether the artwork layout calls for any color mixing wells that would require two templates in a single reaction at carefully balanced ratios. Once those details are locked in, this backbone composition should give every well a fair shot at bright, stable signal across the full 36 hour incubation.

4. The final phase of this lab will be analyzing the fluorescence data we collect to determine whether we can draw any conclusions about favorable reagent compositions for our fluorescent proteins. This will be due a week after the data is returned (date TBD!). The reaction composition for each well will be as follows:

6 μL of Lysate 10 μL of 2X Optimized Master Mix from above 2 μL of assigned fluorescent protein DNA template 2 μL of your custom reagent supplements Total: 20 μL reaction Here is how I would think about designing the 2 µL custom supplement spike in given this fixed reaction architecture, since that small volume is effectively your only experimental lever once the lysate, master mix, and DNA template are locked in.

The key constraint is that your 2 µL supplement represents only 10 percent of the final reaction volume, so any reagent you add needs to be prepared as a 10X concentrated stock to reach its intended working concentration in the final 20 µL. For example, if you want final 100 µM bilirubin to support an Electra2 well, your supplement stock needs to be 1 mM bilirubin, and if you want a final 2 mM additional HEPES-KOH pH 7.5 on top of what the master mix already provides, your stock needs to be 20 mM. This 10X rule is the single most important arithmetic check before pipetting anything, because a concentration error here propagates directly into your fluorescence readout and muddies any conclusions you try to draw later.

Given the acid sensitivity and maturation kinetics concerns from the earlier analysis, I would design the 2 µL custom supplement as a targeted cocktail rather than a single reagent, so you get maximum information from each well. A strong candidate composition would be 20 mM HEPES-KOH pH 7.5 (for 2 mM final buffer boost to resist acidification), 500 µM bilirubin if you are assigned an Electra2 well (for 50 µM final, since Electra2 fluorescence is bilirubin limited in lysate), 10 mM putrescine (for 1 mM final, to stabilize ribosomes over 36 hours), and 10 mM DTT (for 1 mM final, to keep cysteine residues reduced and protect the chromophore environment in oxygen sensitive proteins). If you are working with mKO2, mRFP1, or mScarlet-I, the HEPES boost is the most important ingredient; if you have Electra2, bilirubin is non negotiable; and if you have sfGFP or mTurquoise2, you can probably skip the bilirubin and use that volume budget for slightly more buffer or a small magnesium bump.

For experimental design and downstream analysis, the smart move is to coordinate with classmates so that different wells carry different supplement compositions in a systematic way, which converts the collaborative painting into a small factorial experiment. For instance, if three wells all express mKO2 but one receives a HEPES boost, one receives a DTT boost, and one receives both, you can directly compare endpoint fluorescence and kinetic curves to isolate which intervention mattered most. Without that coordination, every well becomes a one off anecdote and the fluorescence data will be hard to interpret beyond “this well was brighter than that one.” Before the data arrives, it is worth drafting a simple spreadsheet listing each well, its assigned protein, its supplement composition, and the hypothesis you are testing, because that document becomes the backbone of your final analysis writeup.

For the analysis phase itself, the core questions to answer from the fluorescence time course data are: (1) did the supplement move the endpoint brightness up, down, or not at all relative to control wells, (2) did the supplement change the shape of the kinetic curve (faster rise, later plateau, less decline after hour 20), and (3) does the effect size depend on which fluorescent protein was expressed, which would confirm or refute the protein specific hypotheses about acid sensitivity, oxygen dependence, and maturation kinetics. Plotting normalized fluorescence versus time for each supplement condition, overlaid by protein, will make patterns jump out far faster than staring at endpoint numbers alone, and fitting a simple logistic or Gompertz growth curve to each trace gives you quantitative parameters (maximum signal, half time, decay rate) you can compare statistically across conditions. Once those numbers are in hand, the writeup essentially writes itself: state the hypothesis, show the curve, report the fitted parameters, and comment on whether the biophysical property you targeted actually explains the observed effect.

Part D: Build-A-Cloud-Lab | (optional) Bonus Assignment

Use this simulation tool to create an interesting looking cloud lab out of the Ginkgo Reconfigurable Automation Carts. This is just a minimal implementation so far, but I would love to see some fun designs!