Week z11 HW: Bioproduction and Cloud Labs

Part A: Pixel Art

Contribute at least one pixel to this global artwork experiment before the editing ends on Sunday 4/19 at 11:59 PM EST. Make a note on your HTGAA webpages.

I liked that the project setup limited the amount of changes that each user could make at once (with the time buffer) to ensure contribution from many users and to reduce the chaos of changes happening at once. In terms of potential improvements, the size of the canvas (/total number of plates) could be increased for next year to increase the number and complexity of possible designs.

Part B: Cell-Free Synthesis

Referencing the cell-free protein synthesis reaction composition, provide a 1-2 sentence description of what each component’s role is in the cell-free reaction.

E coli lysate:

  • BL21 Star Lysate w/ T7 Polymerase: E. coli minimal cell material that provides a basis/medium for all the reactions taking place and is often optimized for minimal mRNA and protein degradation (via reduced RNase and protease activity). The T7 polymerase synthesizes RNA for in vitro transcription.

Salts Buffer

  • Potassium Glutamate: stabilizes RNA and aids in protein folding and interactions via charge
  • HEPES-KOH pH 7.5: acts as a buffer to maintain optimal pH for “cell” functioning
  • Magnesium Glutamate: provides a source of Mg2+, an essential cofactor for many enzymes invovled in transcription and translation – e.g., RNA polymerases and ATPases
  • Potassium phosphate monobasic: another buffer component to maintain optimal pH. Also provides phosphate ions essential for ATP-regeneration and other cellular processes.
  • Potassium phosphate dibasic: similar role to potassium phosphate monobasic, the key difference being that the dibasic version is an alkaline buffering agent while monobasic is an acidic buffering agent. At the correct concentrations, the two together help create the optimal pH.

Energy / Nucleotide System

  • Ribose: An essential component of ATP and a precursor to deoxyribose found in DNA and RNA
  • Glucose: Essential for ATP production (via glycolysis)
  • AMP: Second messenger for various essential processes and a precursor to ATP. Also a nucleic acid monomer
  • CMP: Nucleic acid monomer and essential for RNA synth
  • GMP: Also a nucleic acid monomer
  • UMP: RNA monomer
  • Guanine: Its derivatives are essential for RNA synthesis

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: Provides amino acids essential for translation/protein synth
  • Tyrosine: Not present in the standard mix because it’s considered “non-essential” because it is synthesized from the already-provided phenylalanine precursor, but is important to provide to ensure sufficient supply
  • Cysteine: Not present in the standard mix because it’s considered “non-essential” because it is synthesized from the already-provided phenylalanine precursor, but is important to provide to ensure sufficient supply

Additives

  • Nicotinamide: Precursor to NAD+, essential for ATP synthesis

Backfill

  • Nuclease Free Water: Suspends the solution so that everything is mixed together and can function in a cell-like manner (cells are ~70% water)

Part C: 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)

  2. sfGFP: Has “superfolder” mutations that give it a stronger beta barrel and enhanced folding resilient to sub-optimal conditions

  3. mRFP1: Unlike most fluorescent proteins, mRFP1 is monomeric, so can be easily fused to other proteins

  4. mKO2: Is also monomeric and is engineered with mutations that enhance its folding speed and stability and reduce auto-aggregation

  5. mTurquoise2: Also monomeric and has a strong “packed” structure around the chromophore (due to a key AA substitution) that enhances its stability and ability to withstand photobleaching

  6. mScarlet_I: Also monomeric and has an especially rigid chromophore structure that enhances its brightness.

  7. Electra2: Also monomeric and has a tight beta barrel around its chromophore that strengthens its general stability and photostability.

  8. 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.

Hypothesis: Increasing the concentrations of Potassium Glutamate and Magnesium Glutamate will enhance the stability (and fluoresence, which depends on correct folding) of the beta barrel-based proteins above (e.g., sfGFP).

  1. 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.

  2. 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