Week 11 HW: Bioproduction & Cloud Labs

Week 11 — Bioproduction & Cloud Labs

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

Make a note on your HTGAA webpages including:

(a) What you contributed to the community bioart project (e.g., “I made part of the DNA on the bottom right plate”)

Honestly, I didn’t get to contribute a pixel this time. The window between the personalized URL going out and the editing deadline on Sunday 4/19 closed before I was able to sit down and place mine, which I’m a bit bummed about because the project sounded cool.

(b) What you liked about the project

There’s something charming about turning a piece of lab plasticware into a collaborative painting. The fact that the “paint” is actually six different fluorescent proteins (so the colours come from real biology, not a filter) made it feel meaningful in a way that ordinary digital art wouldn’t. And the global participation aspect was the part I kept thinking about: strangers in different time zones jointly producing one coherent image is exactly the kind of thing cloud labs are supposed to make possible.

(c) What about this collaborative art experiment could be made better for next year

The biggest thing for me would be a longer contribution window.

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

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 with T7 RNA Polymerase: This is the engine of the whole reaction. Lysing the cells gives you all the ribosomes, tRNAs, translation factors, and aminoacyl-tRNA synthetases you need, and the BL21 Star background means RNase E is knocked down so your transcripts last longer. The included T7 RNAP transcribes any DNA template that’s under a T7 promoter.

Salts / Buffer

• Potassium glutamate: The main monovalent cation source. Glutamate is used instead of chloride because it’s much gentler on translation. Chloride tends to inhibit ribosome activity at the concentrations you’d need.
• HEPES-KOH pH 7.5: Buffers the reaction near physiological pH. This matters a lot over long incubations because cell-free reactions tend to acidify as energy substrates get metabolized.
• Magnesium glutamate: Mg²⁺ is essential for ribosome assembly, RNAP activity, and basically every NTP-using enzyme. The concentration is fussy: too little and translation stalls, too much and you get misreading.
• Potassium phosphate (mono- and dibasic): Together they act as a secondary buffer and supply inorganic phosphate, which feeds back into NTP regeneration.

Energy / Nucleotide System

• Ribose: Substrate for the pentose phosphate pathway in the lysate; gets converted into PRPP, which is used to build nucleotides from free bases.
• Glucose: Cheap, slow-burn energy source feeding glycolysis. It produces ATP gradually rather than all at once, which is what makes the long-incubation format possible.
• AMP, CMP, GMP, UMP: The NMPs are starting material for nucleotide regeneration. Endogenous kinases in the lysate phosphorylate them up to NTPs, which are what RNAP uses.
• Guanine: Free base that feeds into the salvage pathway (more on this in question 3).

Translation Mix (Amino Acids)

• 17 amino acid mix: Provides the pool of monomers for the ribosome to assemble into protein.
• Tyrosine and Cysteine are added separately because they’re less soluble than the others and tend to need their own handling, so they’re broken out of the main mix.

Additives

Nicotinamide: Precursor for NAD⁺/NADH regeneration. The lysate’s redox metabolism eats through these cofactors quickly and keeping the NAD pool topped up helps maintain energy regeneration over long reactions.

Backfill

Nuclease-Free Water: Brings the reaction to its final volume without introducing contaminating RNases or DNases that would chew up your template or transcripts.

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 is built for speed: it ships pre-formed NTPs and uses phosphoenolpyruvate as a high-energy phosphate donor, so ATP regeneration happens in basically a single enzymatic step (via pyruvate kinase). You get a fast burst of expression, but PEP is expensive and gets exhausted quickly, so the reaction plateaus within an hour.

The 20-hour NMP-Ribose-Glucose mix takes the opposite approach. Instead of pre-built NTPs, it relies on the lysate’s own glycolysis and pentose phosphate pathway to slowly assemble NTPs from cheaper precursors (NMPs, ribose, glucose). Yields per unit time are lower but the reaction sustains itself for much longer.

In practice, PEP-NTP is what you’d reach for in a screen where you want a quick yes/no on expression, while NMP-Ribose-Glucose is the right call for our 36-hour fluorescence experiment where total integrated signal matters more than how fast it gets there.

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

Through the purine salvage pathway. The lysate retains the enzymes that normally let E. coli recycle bases rather than building them from scratch. Free guanine gets joined to PRPP (5-phosphoribosyl-1-pyrophosphate, made from the ribose in the energy mix) by the enzyme HGPRT, producing GMP. From there, guanylate kinase phosphorylates GMP → GDP, and nucleoside diphosphate kinase finishes the job → GTP, which is the actual substrate T7 RNAP uses for transcription. So even without GMP in the mix directly, the reaction generates it on demand, which is partly why ribose is included in the energy system in the first place.

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. (1-2 sentences each)

Hint: options include maturation time, acid sensitivity, folding, oxygen dependence, etc.

sfGFP (superfolder GFP): The standout property here is folding robustness. sfGFP was engineered specifically to fold correctly even from poorly behaved fusion partners or under suboptimal conditions. In a cell-free lysate, where chaperone availability is limited compared to a live cell, this matters a lot: you get reliable maturation and a clean fluorescence readout even if other reaction parameters drift.

mRFP1: Slow chromophore maturation is the big one. mRFP1 was an early-generation monomeric red derived from DsRed, and while it fixed the tetramerization problem, the maturation half-time is still on the order of an hour, meaning a chunk of synthesized protein in a 36-hour cell-free run will be sitting in a non-fluorescent intermediate state, especially at the early time points.

mKO2: Acid sensitivity. mKO2 has a relatively high pKa (~5.5), which sounds fine in isolation, but cell-free reactions noticeably acidify over long incubations as glycolytic byproducts accumulate. This means mKO2’s apparent brightness can drop later in the run not because less protein is being made, but because more of it is sitting in a protonated, dim state.

mTurquoise2: Very high quantum yield (~0.93) and tight folding kinetics. The practical consequence in cell-free is that you get an unusually favorable signal-to-noise ratio per molecule of folded protein, so mTurquoise2 is forgiving of low expression yields. It’s also a popular FRET donor for the same reason.

mScarlet-I: The “I” variant was engineered specifically for faster maturation than the original mScarlet, trading a small amount of brightness for speed. In a 36-hour cell-free run that distinction shows up clearly: you see signal accumulation earlier in the time course rather than only at late time points, which is the main reason mScarlet-I tends to be preferred over mScarlet in dynamic measurements.

Electra2: Oxygen-dependent chromophore maturation. All FPs in the GFP/DsRed family need molecular O₂ for the autocatalytic cyclization step that forms the fluorophore, but Electra2 is on the more demanding end. In a sealed or partially anaerobic cell-free reaction, that can cap the fraction of molecules that ever become fluorescent.

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.

I’m going to base my hypothesis on sfGFP, partly because it’s the protein I have the cleanest mental model for and partly because its main bottleneck is something you can actually push on with the master mix.

Hypothesis: For sfGFP, modestly increasing magnesium glutamate (in the 8 to 12 mM range) together with topping up the energy regeneration components, specifically ensuring sustained NTP supply via the NMP-Ribose-Glucose route, will increase the time-integrated fluorescence over a 36-hour incubation.

Reasoning: sfGFP’s folding is essentially not the rate-limiting step (that’s the whole point of “superfolder”), so the real ceiling on its 36-hour signal is how much protein the system can actually translate before energy or ribosome activity gives out. Mg²⁺ directly affects both ribosome stability and translation fidelity, so a small bump should improve elongation rates without tipping into misreading territory. And because sfGFP matures fast and is photostable, every additional molecule synthesized translates almost immediately into detectable signal, with no maturation backlog masking the gains.

Expected effect: Earlier rise to a higher fluorescence plateau, and crucially a flatter decay curve toward the back end of the 36-hour window, since translation continues feeding new molecules into a pool that doesn’t lose signal quickly.

Notes on next phases

The second phase of this lab will be to define the precise reagent concentrations for the cell-free experiment. Wells with specific fluorescent proteins will be assigned and instructions sent by email by April 24. Master mix compositions can be drafted ahead of time.

The final phase will be analyzing the fluorescence data once it comes back, to see if any conclusions can be drawn about favorable reagent compositions for our fluorescent proteins. Due a week after the data is returned (date TBD).

Reaction composition per well