Week 11 HW: Bioproduction & Cloud Labs

Part A — The 1,536 Pixel Artwork Canvas

Unfortunately, I wasn’t able to contribute to the pixel artwork before the April 19 deadline. Looking at the final result, though, I found the concept of emergent collective creativity really compelling… hundreds of independent decisions producing a coherent image is a great parallel to how biological systems self-organize. For next year, a more prominent reminder with a countdown timer on the course page itself would help students like me who missed the email with the personalized URL. It would also be interesting to have a live preview of the canvas as it fills up so contributors can make more intentional decisions about where their pixels fit into the bigger picture.

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

1. Role of each component:

E. coli Lysate — BL21 (DE3) Star Lysate (includes T7 RNA Polymerase)

This is the core of the entire reaction. It provides all the molecular machinery needed to go from DNA to protein, including ribosomes, translation factors, chaperones, and T7 RNA polymerase. The BL21 (DE3) Star strain is particularly well-suited because the “Star” mutation disables RNase E, slowing mRNA degradation and allowing sustained protein production over longer reaction times.

Potassium Glutamate

Potassium ions are essential for ribosome stability and activity. Glutamate is used as the counterion instead of chloride because chloride inhibits transcription and translation at the concentrations needed, while glutamate is metabolically compatible and doesn’t interfere with the reaction machinery.

HEPES-KOH pH 7.5

This is the main buffer of the reaction. As transcription and translation proceed, acidic byproducts accumulate and the pH drops, which disrupts ribosome function. HEPES maintains a stable pH around 7.5, keeping conditions optimal for both the RNA polymerase and the ribosome throughout the reaction.

Magnesium Glutamate

Magnesium is one of the most critical ions in the system. Ribosomes require it as a structural cofactor, RNA polymerase depends on it catalytically, and ATP functions primarily as an Mg-ATP complex in enzymatic reactions. The concentration needs to be carefully optimized because too little stops the reaction and too much causes inhibition and precipitation.

Potassium Phosphate Monobasic / Dibasic

Together these form a secondary phosphate buffer that adds pH stability on top of HEPES. They also supply inorganic phosphate that feeds into energy regeneration reactions happening within the lysate, since phosphate is both a substrate and product of ATP metabolism.

Ribose

Ribose feeds into the pentose phosphate pathway enzymes present in the lysate, supporting NADPH regeneration and nucleotide biosynthesis. It’s a key part of what makes this a long-duration energy system. Rather than being a one-shot phosphate donor, it continuously supports metabolism throughout the reaction.

Glucose

Glucose is the primary carbon and energy source for the 20-hour reaction format. The glycolytic enzymes in the lysate metabolize it to pyruvate, regenerating ATP in the process. Unlike simpler systems that rely on a fixed phosphate donor like PEP, glucose provides sustained energy by essentially running a simplified version of central carbon metabolism inside the reaction tube.

AMP, CMP, GMP, UMP

These nucleoside monophosphates are the precursors for RNA synthesis. Rather than adding costly NTPs directly, the system provides monophosphate forms that get phosphorylated to triphosphates by kinases already present in the lysate. This approach avoids the transcriptional inhibition that can come from high NTP concentrations and makes the system significantly more economical for long reactions.

Guanine

Guanine base is added separately because the guanine nucleotide pool depletes faster than other nucleotides during extended reactions. GTP is consumed heavily both as a transcription substrate and as an energy carrier during translation elongation. Adding free guanine allows the lysate’s nucleotide salvage pathways to continuously replenish GTP, preventing it from becoming a bottleneck.

17 Amino Acid Mix

This provides 17 of the 20 standard amino acids needed for protein synthesis. Tyrosine and cysteine are left out of this mix and added separately because they have poor solubility or stability under standard storage conditions (tyrosine is nearly insoluble at neutral pH and cysteine oxidizes readily, so both need to be prepared and handled differently).

Tyrosine

Tyrosine is added as a separate component because it has very low solubility at neutral pH and can’t be included in a standard amino acid mix without precipitation issues. It’s essential for translation though, since many proteins including fluorescent proteins rely on tyrosine for their chromophore formation.

Cysteine

Cysteine is kept separate because it oxidizes easily and can form disulfide bonds with other cysteines in the mix, depleting the free amino acid pool before it ever gets incorporated into protein. Adding it fresh and separately ensures it’s available in its reduced, usable form during translation.

Nicotinamide

Nicotinamide is a precursor to NAD+ and NADP+, which are essential cofactors for many of the oxidoreductase reactions running in the lysate during energy metabolism. Without replenishing these cofactors, the redox balance in the reaction shifts and energy regeneration slows down. Including nicotinamide helps maintain the NAD+/NADH pool throughout the reaction.

Nuclease Free Water

This is the backfill, essentially used to bring the reaction up to its final volume after all other components have been added. Using nuclease-free water is important because even trace amounts of RNases or DNases would degrade your mRNA or DNA template and kill the reaction.

2. Differences between the 1-hour PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix:

The main difference comes down to how each system generates and sustains the energy needed for transcription and translation. The 1-hour PEP-NTP system is a fast, straightforward approach. Phosphoenolpyruvate acts as a direct phosphate donor to regenerate ATP from ADP through pyruvate kinase, and pre-formed NTPs are provided directly for transcription. It works quickly but burns through its substrates fast, making it suitable only for short reactions. The 20-hour NMP-Ribose-Glucose system takes a completely different approach. It provides nucleoside monophosphates instead of triphosphates, and uses glucose and ribose as carbon sources that feed into glycolysis and the pentose phosphate pathway to continuously regenerate both ATP and NTPs from within the lysate’s own metabolism. This makes it far more self-sustaining, but it requires more complex metabolic activity from the lysate and takes longer to ramp up. Essentially the PEP-NTP system is optimized for speed and simplicity while the NMP-Ribose-Glucose system is optimized for yield and duration.

3. How can transcription occur if GMP is not included but Guanine is?

When you add free guanine base to the reaction, the lysate’s nucleotide salvage pathway enzymes convert it back into GMP, then GDP, and finally GTP through a series of phosphorylation steps using ATP as the phosphate donor. Specifically, hypoxanthine-guanine phosphoribosyltransferase (HGPRT) converts guanine to GMP using PRPP (phosphoribosyl pyrophosphate) as the ribose-phosphate donor, and then guanylate kinase and nucleoside diphosphate kinase phosphorylate it up to GTP. So you’re not skipping GMP, you’re just letting the lysate make it itself from the free base, which is actually more efficient because free bases are cheaper and more stable than nucleotides.

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

1. Biophysical or functional properties of each fluorescent protein:

sfGFP

sfGFP (superfolder GFP) was specifically engineered to fold robustly even when fused to poorly folding proteins. Its key property in cell-free systems is its extremely fast and reliable folding , it reaches full fluorescence quickly after synthesis, making it a great positive control. The one caveat is that like all GFP variants, it requires molecular oxygen for chromophore maturation, so in reactions where oxygen is limited (deep in a well plate under a seal), you might see lower fluorescence than expected even if protein yield is high.

mRFP1

mRFP1 was the first true monomeric red fluorescent protein, derived from DsRed. Its main limitation in cell-free systems is relatively slow chromophore maturation — it takes significantly longer than GFP variants to become fully fluorescent after being synthesized. This means in a short reaction window you might underestimate how much protein was actually made, and for a 36-hour incubation you need to account for the fact that fluorescence will keep increasing even after active translation has stopped. It also has lower brightness than newer red variants.

mKO2

mKO2 is a monomeric orange fluorescent protein derived from Kusabira Orange. Its key biophysical property relevant to cell-free expression is that it has a relatively long maturation time , longer than GFP but slightly better than mRFP1. It’s also pH-sensitive, with fluorescence decreasing noticeably below pH 6. In a cell-free reaction where pH can drift as acidic byproducts accumulate, this pH sensitivity could cause you to underestimate actual protein concentration if buffering isn’t maintained well throughout the reaction.

mTurquoise2

mTurquoise2 is one of the best cyan fluorescent proteins available , it has an exceptionally high quantum yield and is one of the brightest proteins in the cyan range. For cell-free systems its main advantage is fast maturation and high photostability, making it ideal for long 36-hour reads where you need reliable signal over time. One thing to watch out for is spectral bleed-through into the GFP channel if you’re running a multiplexed reaction, since its emission tail overlaps with sfGFP excitation.

mScarlet-I

mScarlet-I is a fast-maturing variant of mScarlet, engineered specifically to improve maturation speed over the original while maintaining high brightness. In cell-free systems this fast maturation is its biggest advantage , you can get reliable fluorescence readout much earlier in the reaction compared to mRFP1 or mKO2. It’s also relatively insensitive to pH in the physiological range, which makes it more robust in cell-free conditions where pH management isn’t perfect.

Electra2

Electra2 is a relatively new infrared-range fluorescent protein. Its key property that matters in cell-free systems is that it requires a biliverdin chromophore cofactor that is not naturally present in E. coli lysate unlike GFP-based proteins that autocatalytically form their chromophore from their own amino acids, Electra2 needs exogenous biliverdin added to the reaction to fluoresce at all. This makes it uniquely challenging in cell-free systems , if you don’t supplement the reaction with biliverdin, you’ll get zero fluorescence even if the protein is being made perfectly well.

2. Hypothesis for improving fluorescence over 36-hour incubation:

I’m focusing on Electra2 since it has the most obvious and addressable limitation in cell-free conditions.

Hypothesis: Supplementing the cell-free master mix with exogenous biliverdin at a concentration of 25–50 μM will significantly increase Electra2 fluorescence output over a 36-hour incubation compared to unsupplemented reactions.

The reasoning is straightforward: Electra2 is a biliverdin-dependent fluorescent protein, meaning it can’t form a functional chromophore without this cofactor. E. coli lysate contains no meaningful amount of biliverdin because bacteria don’t have the heme oxygenase pathway that produces it in mammalian cells. So no matter how well the protein folds or how much of it gets made, none of it will be fluorescent without biliverdin present. By adding biliverdin directly to the custom reagent supplement slot in the 2 μL addition, every newly synthesized Electra2 molecule will immediately have access to its chromophore precursor, maximizing the fraction of protein that becomes fluorescent. The expected effect is a large increase in fluorescence signal, essentially “unlocking” the protein’s fluorescence that would otherwise be completely invisible. A titration of biliverdin concentration (0, 10, 25, 50, 100 μM) would let you find the optimal amount without wasting cofactor or potentially causing any inhibitory effects at very high concentrations.

3. Master Mix Compositions:

My 8 well assignments and their custom reagent adjustments are as follows:

Q1-D19 — Electra2 (Low energy condition): Default composition with glucose increased slightly above baseline to provide a modest boost in sustained energy metabolism.

Q1-E19 — Electra2 (Medium energy condition): Glucose increased moderately above baseline, ribose increased once above baseline to support both glycolysis and the pentose phosphate pathway simultaneously.

Q1-F19 — Electra2 (High energy condition): Glucose increased substantially above baseline, ribose increased twice above baseline, AMP increased once to provide additional nucleotide precursors for sustained transcription.

Q2-A1 — mRFP1 (Low magnesium boost): Magnesium Glutamate increased twice above baseline to modestly enhance ribosome activity and translation speed.

Q2-A2 — mRFP1 (High magnesium boost): Magnesium Glutamate increased four times above baseline to more aggressively test whether higher Mg2+ accelerates maturation.

Q3-H13 — mKO2 (Buffer protection): HEPES-KOH increased twice above baseline to maintain pH stability throughout the 36-hour incubation and protect mKO2 fluorescence from acid-induced quenching.

Q4-B3 — mTurquoise2 (Amino acid boost): 17 Amino Acid Mix increased once above baseline, Tyrosine increased twice above baseline to address the known solubility limitation of tyrosine in standard cell-free amino acid mixes.

Q1-D1 — mScarlet-I (Sustained energy): Glucose increased twice above baseline, Ribose increased once above baseline, Nicotinamide increased once above baseline to support sustained NAD+ regeneration and energy metabolism over the full reaction duration.