Week 12 HW: Bioproduction and Cloud Labs

The 1,536 Pixel Artwork Canvas | Collective Artwork

I contributed a blue pixel to the bottom right corner. I liked being able to see the project evolve over time. One thing I’d improve for next year is to either make more pixels so people aren’t writing over previously drawn pixels or to limit the number of pixels someone can add and see how a collective artwork emerges that way.

Cell-Free Protein Synthesis | Cell-Free Reagents

E. coli Lysate

  • BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): contains all the cellular machinery to complete transcription and translation (polymerase, ribosome, tRNAs, etc.)

Salts/Buffer

  • Potassium Glutamate: stabilizes protein folding, facilitates ribosome function
  • HEPES-KOH pH 7.5: maintains pH
  • Magnesium Glutamate: ribosome structure (and RNA folding), tRNA charging, etc.
  • Potassium phosphate monobasic: pH stabilization, phosphate source
  • Potassium phosphate dibasic: pH stabilization, phosphate source

Energy / Nucleotide System

  • Ribose: regenerate nucleotides and make ATP
  • Glucose: make ATP
  • AMP: used to synthesize/make up DNA
  • CMP: used to synthesize/make up DNA
  • GMP: used to synthesize/make up DNA
  • UMP: used to synthesize/make up DNA
  • Guanine: used for extra GTP required by translation

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: necessary for making up the protein
  • Tyrosine: necessary for making up the protein, separate because of solubility issues
  • Cysteine: necessary for making up the protein, separate because reactive (oxidizes)

Additives

  • Nicotinamide: precursor to NAD+/NADP+

Backfill

  • Nuclease Free Water: ensure everything reaches the correct concentration

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 master mix lacks some pH stabilizing salts, and uses a different carbon source. It also uses different additives. Also, the nucleotides are already tri-phosphorylated, rather than just having one phosphate.

Planning the Global Experiment | Cell-Free Master Mix Design

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)

  • sfGFP: matures quickly, allowing for quick fluorescence after synthesis
  • mRFP1: matures slowly, so it may not fluoresce as brightly, but is not very sensitive to acid, making it resistant to pH changes
  • mKO2: somewhat sensitive to acid changes, potentially causing it to not fluoresce as brightly in suboptimal conditions
  • mTurquoise2: matures quickly and is not sensitive to acid, allowing it to fluoresce quickly and brightly in many varying pH
  • mScarlet_I: somewhat sensitive to acid changes, potentially causing it to not fluoresce as brightly in suboptimal conditions
  • Electra2: oxygen dependent, so it may not function properly in poorly-oxygenated conditions

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, perhaps adding chaperone proteins to help it fold could stabilize its fold, and ensuring that the pH does not change much during the incubation using a buffer would help.