week 11 HW: Building Genomes

Question 1: Component Roles in the NMP-Ribose Cell-Free Reaction

E. coli Lysate — BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) This is the core catalytic engine of the reaction — it provides ribosomes, translation factors, chaperones, tRNA synthetases, and metabolic enzymes needed for protein synthesis. The BL21 (DE3) strain specifically expresses T7 RNA Polymerase, which is required to transcribe genes under T7 promoter control.

Salts/Buffer

  • Potassium Glutamate: Provides the primary ionic environment for the reaction, mimicking intracellular potassium concentrations that support ribosome stability and translation fidelity.
  • HEPES-KOH pH 7.5: Maintains a stable physiological pH throughout the reaction, preventing enzyme inactivation and ensuring optimal ribosome function.
  • Magnesium Glutamate: Magnesium is critical for ribosome assembly and stability, as well as for stabilizing nucleotide triphosphates used in transcription and translation.
  • Potassium Phosphate monobasic/dibasic (1.6:1): Acts as a secondary buffer and provides inorganic phosphate, which is important for nucleotide regeneration and energy metabolism in the ribose-based system.

Energy / Nucleotide System

  • Ribose: The primary carbon and energy source in this system — cellular enzymes in the lysate convert ribose into nucleotides and ATP through salvage and phosphorylation pathways, enabling sustained long-duration reactions.
  • Glucose: Supplements ribose as an additional carbon source and feeds into glycolytic pathways to help regenerate ATP and support metabolic activity.
  • AMP, CMP, UMP: Nucleoside monophosphates that serve as precursors for the four RNA nucleotides needed for transcription; the lysate’s kinases phosphorylate these to their triphosphate forms using energy from ribose/glucose metabolism.
  • GMP: Listed at 0 µM in this formulation, suggesting guanine nucleotides are supplied sufficiently via the guanine base and salvage pathway instead.
  • Guanine: A nucleobase that feeds into the purine salvage pathway to support GTP synthesis, complementing the NMP-based nucleotide supply strategy.

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: Supplies the majority of the 20 standard amino acids needed for ribosomal translation of the target protein.
  • Tyrosine pH 12: Tyrosine has very low solubility at neutral pH, so it is dissolved at pH 12 and added separately to avoid precipitation in the master mix.
  • Cysteine: Also added separately because it is redox-sensitive and can oxidize rapidly, which would reduce its availability for translation and potentially disrupt protein folding.

Additives

  • Nicotinamide: A precursor to NAD⁺, which is required as a cofactor for many of the metabolic enzymes in the lysate that regenerate energy from ribose and glucose. Supporting NAD⁺ levels helps sustain the long 20-hour reaction.

Backfill

  • Nuclease-Free Water: Used to bring the reaction to its final volume without introducing RNases or DNases that would degrade the mRNA transcript or DNA template and kill the reaction.

Question 2: Differences between the 1-hour PEP-NTP and 20-hour NMP-Ribose master mixes

The most fundamental difference is in how energy and nucleotides are supplied. The 1-hour PEP-NTP mix provides NTPs (ATP, GTP, CTP, UTP) directly in their fully phosphorylated, ready-to-use form, along with phosphoenolpyruvate (PEP-Mono) as an immediate high-energy phosphate donor for ATP regeneration — this gives fast, high-yield transcription and translation right away but is quickly depleted. The 20-hour NMP-Ribose mix instead provides nucleoside monophosphates and simple sugars (ribose and glucose) as precursors, relying on enzymatic machinery in the lysate to continuously regenerate NTPs from these building blocks, which is slower to ramp up but far more sustainable over longer incubations. The 1-hour mix also contains more additives (spermidine, DMSO, cAMP, NAD, folinic acid) that boost immediate transcription/translation activity, while the 20-hour mix strips these back and instead uses nicotinamide to support the NAD⁺-dependent metabolic activity needed to keep the ribose/glucose energy system running.

Part C: Fluorescent Protein Properties

sfGFP sfGFP (superfolder GFP) is engineered for exceptionally robust folding — it was selected specifically to fold correctly even when fused to aggregation-prone partners, making it highly tolerant of the crowded, non-optimized environment of a cell-free reaction. Its fast and reliable folding makes it an ideal positive control in cell-free systems.

mRFP1 mRFP1 has a relatively slow chromophore maturation time compared to GFP-based proteins, and its fluorescence requires oxidation of the chromophore-forming residues, meaning oxygen availability in the reaction vessel directly limits how much functional protein accumulates over time. In a sealed or low-oxygen cell-free setup, maturation could be significantly delayed.

mKO2 mKO2 is a monomeric orange fluorescent protein with a moderate maturation time, but it is notably sensitive to low pH — its fluorescence decreases substantially below pH 6.5, so maintaining stable buffering throughout a 36-hour reaction is important to preserve signal. It also requires molecular oxygen for chromophore maturation like all GFP-family proteins.

mTurquoise2 mTurquoise2 is one of the brightest and most photostable cyan fluorescent proteins available, with a high quantum yield and fast maturation relative to other cyan variants. However, its emission overlaps with autofluorescence from some cell-free reaction components (particularly NADH), which can complicate quantification at low expression levels.

mScarlet_I mScarlet_I is a fast-maturing red fluorescent protein with high brightness, but like all red FPs it requires sequential cyclization and oxidation steps for chromophore maturation, making it more oxygen-dependent than GFP variants. In long incubation cell-free reactions it performs well once oxygen is available, but early timepoints may underestimate expression.

Electra2 Electra2 is a relatively new far-red fluorescent protein optimized for mammalian expression, and its folding behavior in bacterial cell-free systems is less characterized than the others. Its far-red emission is advantageous for reducing background autofluorescence, but expression yield may be lower in an E. coli lysate context if its folding requirements are not well-matched to the prokaryotic chaperone environment.

Hypothesis for improving fluorescence over 36-hour incubation

Protein: mRFP1 Reagent: Oxygen availability / nicotinamide concentration

Since mRFP1’s chromophore maturation is rate-limited by oxidation, we hypothesize that leaving reactions in a loosely sealed or oxygen-permeable vessel rather than fully sealed, combined with a slightly increased nicotinamide concentration (to sustain NAD⁺-dependent metabolic activity longer), would improve mRFP1 fluorescence over a 36-hour incubation. The increased oxygen exposure would accelerate chromophore maturation, while sustained NAD⁺ levels would keep the ribose/glucose energy system active long enough to continue synthesizing new mRFP1 protein throughout the full reaction window, maximizing total fluorescent protein accumulation.