Week 11 Lab: Cloud Lab

Part B — Cell-Free Protein Synthesis

B1. Role of each reagent (20 h NMP-Ribose-Glucose mix)

B2. PEP-NTP (1 h) vs NMP-Ribose-Glucose (20 h)

The PEP-NTP mix feeds the reaction with finished NTPs and uses phosphoenolpyruvate as a high-energy ATP regenerator — fast, intense protein synthesis, but PEP is depleted within ~1 hour and the system burns out. The NMP-Ribose-Glucose mix instead supplies precursors (NMPs + ribose for PRPP, glucose for glycolytic ATP) and lets the lysate’s own kinases and salvage enzymes assemble NTPs on demand, giving a slower but sustained ramp that lasts 20+ hours. Cost-per-reaction is also much lower because cheap precursors replace expensive NTPs and PEP.

B3. How can transcription occur without GMP if Guanine is present?

E. coli’s purine salvage pathway rebuilds GMP from free Guanine. Specifically:

Ribose -> (ribokinase) -> Ribose-5-P -> (PRPP synthetase) -> PRPP
Guanine + PRPP -> (HGPRT) -> GMP + PPi
GMP -> (GMP kinase) -> GDP -> (NDP kinase) -> GTP

So as long as ribose is supplied (to make PRPP) and HGPRT is active in the lysate, free Guanine gets converted to GTP at a rate that supports transcription. This is the same logic the cell uses to recycle purines released from RNA turnover — Ginkgo / OpenAI’s paper exploited it to cut reagent cost.

Part C — Planning the Global Experiment

C1. Biophysical / functional property of each FP that matters for cell-free expression

a. sfGFP (superfolder GFP) — Engineered for robust, fast folding (~13 min maturation) even when fused to aggregation-prone partners. Chromophore (Ser-Tyr-Gly cyclization -> dehydration -> oxidation) requires O2, but maturation is fast enough that O2 rarely limits it in 20 uL wells.

b. mRFP1 — Classic monomeric DsRed-derivative. Slow maturation (~1 h) through a GFP-like green intermediate before red, O2-dependent, and acid-sensitive (pKa ~4.5). Maturation kinetics, not synthesis rate, dominate readout over a 36 h incubation.

c. mKO2 — From Fungia concinna. Acid-stable (pKa ~5.5, lower than most FPs) -> fluorescence is preserved as glycolysis acidifies the well over long incubations. Maturation is fast (~7 min). Bright per molecule.

d. mTurquoise2 — Engineered CFP variant with exceptional quantum yield (~0.93) and long fluorescence lifetime -> very bright per folded molecule. Folding is reliable, but it shares the standard O2 dependence and emits in cyan, requiring the right filter set.

e. mScarlet-I — De novo designed monomeric RFP, “I” = improved maturation (~36 min, vs ~3 h for parent mScarlet). Among the brightest monomeric reds. Acid-sensitive (pKa ~5.4) and O2-dependent — maturation rate is the key bottleneck and matches the 36 h incubation window well.

f. Electra2 — Engineered FP designed for improved photostability / brightness. As with most FPs, chromophore maturation is O2-dependent and folding efficiency at 37 C will set how much of the synthesized protein actually fluoresces; if it’s a slower-folding variant, longer incubation favors it.

C2. Hypothesis

Protein: mScarlet-I (red). Reagent change: Increase Mg 2+ glutamate from 7 mM to ~10 mM AND extend incubation in mild orbital shaking instead of static. Expected effect:

1. Moderately higher Mg 2+ improves T7 RNAP processivity and ribosome activity -> more mScarlet-I polypeptide synthesized within the first 6 h.
2. Gentle shaking keeps dissolved O2 saturated in the 20 uL droplet, accelerating mScarlet-I’s O2-dependent chromophore oxidation -> more of the synthesized polypeptide reaches the fluorescent state by 36 h.
3. Net: higher endpoint fluorescence at 36 h vs the control mix.

Risks: Too much Mg 2+ causes misincorporation and aggregation; shaking can foam the lysate. Sweep Mg 2+ in 1 mM increments around 10 mM to find the optimum.

C3. Phase 2 — assigned wells & master mix recipe

Wait for assignment email (by 4/24). Once received, fill in:

• Assigned well(s): [e.g., row C col 7, mScarlet-I template]
• Custom 2 uL supplement composition (final concentrations after 1:10 dilution into the 20 uL reaction):
  • [reagent 1: target conc]
  • [reagent 2: target conc]
  • [backfill: nuclease-free water]

Reaction recipe per well (20 uL total):