week-11-hw-building-genomes

Bioproduction & Cloud Labs

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

My Contribution

I contributed pixels forming part of the DNA helix structure on the lower left quadrant of the collective canvas, using a blue-green palette consistent with the biological theme of the artwork.

What I Liked

The most compelling aspect of this project was how it translated the logic of collaborative biology into a visual format. Just as no single cell produces an organism, no single contributor produced the artwork — the final image only emerged through collective action across dozens of participants working asynchronously. There is something genuinely elegant about using a 1,536-well plate format, the same format used for high-throughput biological screening, as the canvas unit. It collapsed the boundary between the scientific instrument and the artistic medium in a way that felt intentional rather than gimmicky.

What Could Be Improved

The main friction point was the personalized URL system. If a participant missed the email or their Discourse account was not linked correctly, there was no clear fallback to still contribute. For next year, it would be worth building a redundant access pathway so that every enrolled student can contribute regardless of email delivery issues. Additionally, allowing contributors to see a live preview of the growing artwork in real time rather than waiting for the editing window to close would significantly increase engagement and give participants a stronger sense of their individual impact on the collective piece.

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

Component Roles in the Cell-Free Reaction

E. coli Lysate — BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) The lysate provides the complete molecular machinery required for gene expression, including ribosomes, translation factors, RNA polymerase (T7 RNAP for driving expression from T7 promoters), chaperones, and metabolic enzymes. It is the functional core of the cell-free system, supplying all the biological components that would normally be found inside a living cell.

Potassium Glutamate Potassium glutamate serves as the primary ionic strength buffer, providing potassium ions that are essential for ribosome stability and translation fidelity. It is preferred over potassium chloride because the glutamate anion is less inhibitory to transcription and translation enzymes.

HEPES-KOH pH 7.5 HEPES is a buffering agent that maintains the reaction pH at 7.5, which is close to the physiological pH of the bacterial cytoplasm and is optimal for ribosome function, RNA polymerase activity, and enzyme catalysis throughout the reaction.

Magnesium Glutamate Magnesium ions are essential cofactors for ribosome assembly, RNA polymerase activity, and ATP hydrolysis. The concentration of magnesium must be carefully titrated as both insufficient and excess magnesium impair translation efficiency.

Potassium Phosphate Monobasic and Dibasic These phosphate salts provide additional buffering capacity and serve as a phosphate source that supports nucleotide regeneration and energy metabolism within the cell-free reaction.

Ribose Ribose is a pentose sugar that serves as a carbon and energy source, feeding into cellular metabolic pathways within the lysate to regenerate nucleoside monophosphates (NMPs) into nucleoside triphosphates (NTPs) needed for ongoing transcription.

Glucose Glucose is a second energy substrate that drives ATP regeneration through glycolytic enzymes retained in the lysate, supplementing ribose to sustain energy supply over longer reaction windows.

AMP, CMP, GMP, UMP These nucleoside monophosphates are the precursors for all four RNA bases. In NMP-based systems, they are phosphorylated to their triphosphate forms by kinases present in the lysate, providing the building blocks for mRNA synthesis by T7 RNA polymerase.

Guanine Guanine is a free nucleobase that can be converted to GMP through the purine salvage pathway enzymes retained in the lysate. It provides an alternative route for replenishing the guanosine nucleotide pool without requiring exogenous GMP directly.

17 Amino Acid Mix This mix supplies 17 of the 20 standard amino acids required for ribosomal protein synthesis. Tyrosine and cysteine are excluded from this mix and supplied separately due to their limited solubility and chemical instability under standard preparation conditions.

Tyrosine Tyrosine is supplied separately at pH 12 to maintain its solubility, as it is poorly soluble at neutral pH. It is an essential amino acid for translation and is critical for fluorescent protein chromophore formation in GFP-family proteins.

Cysteine Cysteine is supplied separately because it is chemically reactive and prone to oxidation. It is required for translation of proteins containing cysteine residues and is particularly important for proteins that rely on disulfide bonds or thiol chemistry for function.

Nicotinamide Nicotinamide is a precursor to NAD+ and NADH, supporting cellular redox reactions within the lysate. It helps sustain metabolic activity and energy regeneration over extended incubation periods by maintaining the NAD+/NADH balance needed for glycolysis and other metabolic pathways.

Nuclease Free Water Nuclease-free water serves as the backfill solvent to bring all reactions to their final volume. Using nuclease-free water prevents RNA degradation from RNase contamination, which would otherwise destroy the mRNA template and halt protein synthesis.

Main Differences Between the 1-Hour PEP-NTP and 20-Hour NMP-Ribose-Glucose Systems

The 1-hour PEP/NTP system is optimized for rapid, high-yield protein production by supplying pre-formed nucleoside triphosphates (ATP, GTP, CTP, UTP) and phosphoenolpyruvate (PEP-Mono) as an immediate energy source, enabling fast transcription and translation without requiring the cell to regenerate nucleotides from simpler precursors. In contrast, the 20-hour NMP-Ribose-Glucose system relies on nucleoside monophosphates (AMP, CMP, GMP set at 0 uM with Guanine substituted, UMP) combined with ribose and glucose as simple carbon and energy precursors, which are processed by metabolic enzymes in the lysate to regenerate NTPs sustainably over a much longer window.

The key functional trade-off is longevity versus immediacy: the PEP/NTP system burns through its energy substrates quickly, making it optimal for short-burst expression but poorly suited for reactions requiring sustained protein production beyond one to two hours. The NMP-Ribose-Glucose system sacrifices initial speed for metabolic sustainability, supporting continuous transcription and translation for up to 20 hours by continuously regenerating the nucleotide pool from these simpler upstream precursors. The 20-hour system also uses a simplified additive profile, replacing the spermidine, DMSO, cAMP, NAD, and folinic acid additives of the 1-hour system with just nicotinamide, reflecting its different metabolic strategy for sustaining energy balance over time.

Bonus: How Can Transcription Occur if GMP is Not Included But Guanine Is?

Although GMP is listed at 0.00 uM in the 20-hour NMP-Ribose-Glucose system, transcription can still proceed because guanine is supplied as a free nucleobase at 200 uM. The E. coli lysate retains active purine salvage pathway enzymes, particularly hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which can convert guanine into GMP using phosphoribosyl pyrophosphate (PRPP) as the phosphoribose donor. Subsequent kinases then phosphorylate GMP sequentially to GDP and then GTP, which is the form required by T7 RNA polymerase for incorporation into mRNA. This indirect route allows the system to maintain a functional GTP pool without requiring exogenous GMP, while also enabling tighter control over guanosine nucleotide concentrations by feeding the salvage pathway at the nucleobase level rather than the monophosphate level.

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

Biophysical and Functional Properties of Each Fluorescent Protein

sfGFP (Superfolder Green Fluorescent Protein) sfGFP was engineered with six stabilizing mutations that dramatically improve its folding robustness, allowing it to fold correctly even when expressed as a fusion partner or under suboptimal conditions. In cell-free systems, this enhanced folding efficiency means sfGFP reaches detectable fluorescence faster and at higher yields than wild-type GFP, making it one of the most reliable reporters for cell-free expression experiments.

mRFP1 (Monomeric Red Fluorescent Protein 1) mRFP1 requires molecular oxygen for chromophore maturation, as the oxidation step that generates the red-emitting chromophore from the DsRed-derived scaffold is oxygen-dependent. In cell-free reactions that are run in sealed or oxygen-limited environments, mRFP1 maturation can be rate-limiting and may result in underestimated fluorescence readings relative to the actual amount of protein synthesized.

mKO2 (Monomeric Kusabira Orange 2) mKO2 exhibits a relatively slow chromophore maturation time on the order of several hours at 37°C, which is significant in a cell-free context where the reaction window may be limited. This means fluorescence readout from mKO2 will continue to increase for hours after the reaction has peaked in protein synthesis, making it important to allow sufficient post-synthesis incubation time before measuring fluorescence endpoints.

mTurquoise2 mTurquoise2 has an unusually high quantum yield of approximately 0.93, the highest reported among cyan fluorescent proteins, and a long fluorescence lifetime, making it exceptionally photostable and bright per molecule. In cell-free systems this is advantageous because even modest expression levels produce detectable signal, reducing the pressure on the cell-free reaction to achieve very high protein yields for a useful readout.

mScarlet_I (mScarlet-I) mScarlet-I combines fast chromophore maturation with high brightness, achieving nearly complete maturation within one to two hours at 37°C. Its rapid maturation rate makes it particularly well-suited for cell-free reactions with short incubation windows, as the fluorescence signal more closely tracks the actual rate of protein synthesis in real time rather than lagging behind due to slow chromophore formation.

Electra2 Electra2 is sensitive to acidic pH, with its fluorescence significantly quenched below approximately pH 6.5. In cell-free reactions where metabolic activity can acidify the reaction environment over time as energy substrates are consumed and organic acids accumulate, this pH sensitivity can cause fluorescence to decrease even if the protein itself remains intact and properly folded, making accurate endpoint measurements at long incubation times unreliable without pH monitoring or buffering correction.

Hypothesis for Improving Fluorescence Over 36-Hour Incubation

Target protein: mKO2

Rationale: mKO2 has a slow chromophore maturation time, meaning that a large fraction of the protein synthesized in the early hours of the reaction will not yet be fluorescent when measured at intermediate time points. Over a 36-hour window, maximizing the amount of mKO2 protein synthesized early gives the protein more total time to mature, resulting in higher final fluorescence.

Hypothesis: Increasing the concentration of the 17 Amino Acid Mix, Tyrosine, and Cysteine in the master mix by approximately 1.5-fold above the base 20-hour NMP-Ribose-Glucose concentrations (from 4.10 mM to approximately 6.0 mM for the amino acid mix and tyrosine, and from 4.00 mM to 6.00 mM for cysteine) will increase the rate and total yield of mKO2 translation in the first 6 to 12 hours of the reaction. With a larger pool of synthesized but immature mKO2 protein available at the midpoint of the incubation, the extended 36-hour window provides sufficient time for the slow chromophore oxidation step to proceed to completion, resulting in a higher final fluorescent protein yield than would be achievable in a shorter reaction or with standard amino acid concentrations.

Expected effect: Elevated amino acid concentrations will reduce translational stalling caused by substrate depletion during peak synthesis, front-loading mKO2 production and maximizing the fraction of synthesized protein that has time to mature within the 36-hour window. A potential risk is that excessively high amino acid concentrations could alter ionic strength and compete with magnesium coordination, so careful titration around the 1.5-fold increase range is warranted.

Reaction Composition Reference

Each well reaction will be composed as follows: