Week 11 HW: Building Genomes

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

Somehow I didn’t receive an email, so I couldn’t contribute.

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 Lysate (includes T7 RNA Polymerase):

Salts/Buffer

  • Potassium glutamate: Maintains ionic strength and mimics intracellular conditions, helping stabilize ribosomes and enzymes for efficient translation.
  • HEPES-KOH pH 7.5: Buffers the reaction to keep a stable pH, which is critical for enzyme activity and protein synthesis.
  • Magnesium glutamate: Provides Mg²⁺ ions, essential cofactors for ribosome structure, tRNA binding, and enzymatic reactions in transcription/translation.
  • Potassium phosphate (dibasic/monobasic): Contributes to buffering capacity and supplies phosphate ions needed for energy metabolism and nucleotide balance.

Energy / Nucleotide System

  • Ribose: Serves as a precursor for nucleotide synthesis, enabling regeneration of NTPs required for transcription and energy transfer.
  • Glucose: Acts as an energy source, feeding metabolic pathways that regenerate ATP and drive the reaction.
  • AMP, CMP, UMP: Provide nucleotide building blocks for RNA synthesis and can be converted into triphosphates (ATP, CTP, UTP) for transcription.
  • GMP (0 µM): Its absence suggests reliance on salvage pathways (e.g., from guanine) to generate GTP, a key molecule for translation.
  • Guanine: Precursor for GMP/GTP synthesis via salvage pathways, supporting RNA synthesis and ribosomal function.

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: Supplies the building blocks for protein synthesis (excluding specific ones added separately for stability or solubility reasons).
  • Tyrosine: Added separately due to solubility issues; required as a protein building block once adjusted to a usable form.
  • Cysteine: Included separately because it is prone to oxidation; essential for forming disulfide bonds in proteins.

Additives

  • Nicotinamide: Precursor for NAD⁺/NADH, supporting redox balance and metabolic reactions involved in energy regeneration.

Backfill

  • Nuclease Free Water: Serves as the solvent to bring all components to the desired final volume while preventing degradation of DNA/RNA by nucleases, ensuring the stability of the transcription–translation machinery.

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 optimized PEP–NTP master mix relies on directly supplied high-energy molecules (PEP) and fully formed nucleotides (ATP, GTP, CTP, UTP), enabling rapid and immediate protein synthesis but limiting the reaction’s duration. In contrast, the 20-hour NMP–ribose–glucose system uses metabolic precursors such as NMPs, ribose, and glucose, which are enzymatically converted within the system to regenerate energy and nucleotides over time. This results in a slower initial rate but allows for more sustained, long-term protein production with a simpler and more resource-efficient composition.

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. (Hint: options include maturation time, acid sensitivity, folding, oxygen dependence, etc) (1-2 sentences each)

  1. sfGFP: Superfolder GFP is engineered for robust folding, allowing it to fold efficiently even in challenging conditions like cell-free systems, which leads to strong and reliable fluorescence output.
  2. mRFP1: has a relatively slow maturation time, meaning fluorescence develops more slowly after translation, which can delay signal detection in short experiments. -> low acid sensitivity.
  3. mKO2: is relatively acid-sensitive, so its fluorescence can decrease under lower pH conditions that may arise during longer cell-free reactions.
  4. mTurquoise2: has a high quantum yield and brightness, making it very efficient for fluorescence readout even at lower expression levels. It’s also a rapidly-maturing monomer with very low acid sensitivity.
  5. mScarlet_I: is optimized for fast maturation and high brightness, enabling strong fluorescence signals relatively quickly compared to older red fluorescent proteins.
  6. Electra2: is designed for enhanced brightness and stability, but like many fluorescent proteins, it is oxygen-dependent for chromophore formation, which can limit fluorescence if oxygen is depleted in the system

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.

For mRFP1, which is limited by slow chromophore maturation, increasing oxygen availability in the cell-free mastermix (e.g., by reducing reaction volume-to-surface ratio or incorporating oxygen-rich buffers) and supplementing with energy-regeneration components (e.g., higher glucose or ribose concentrations) will enhance chromophore formation and sustain ATP levels. This will accelerate maturation and prolong protein synthesis, ultimately leading to higher cumulative fluorescence over a 36-hour incubation.

For mKO2, whose fluorescence is acid-sensitive, the 36-hour mastermix could be improved by increasing buffering capacity (e.g., higher HEPES concentration and optimized phosphate ratio) and slightly reducing glucose concentration. In the 20-hour system, glucose metabolism can lead to acidification over time, which would quench mKO2 fluorescence. By strengthening the buffer and limiting excess glucose-driven acid production, the pH can be kept more stable, resulting in higher and more stable fluorescence over a 36-hour incubation.

3. The second phase of this lab will be to define the precise reagent concentrations for your cell-free experiment. You will be assigned artwork wells with specific fluorescent proteins and receive an email with instructions this week (by April 24). You can begin composing master mix compositions here.

4. The final phase of this lab will be analyzing the fluorescence data we collect to determine whether we can draw any conclusions about favorable reagent compositions for our fluorescent proteins. This will be due a week after the data is returned (date TBD!). The reaction composition for each well will be as follows:

6 μL of Lysate
10 μL of 2X Optimized Master Mix from above
2 μL of assigned fluorescent protein DNA template
2 μL of your custom reagent supplements

Total: 20 μL reaction