Week 11 HW: Bioproduction & Cloud Labs

Week 11 — Bioproduction & Cloud Labs

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

Unfortunately, I couldn’t contribute but I think it’s a great project that improves creativity and working in teams. The best part of it is there’s a contribution from all over the world. I think for next year we could have a more detailed explanation of the draw-to-made in order to create something specific but with different points of view. For example to create a plate to draw a bacteria and see what happens. I think this would be interesting.

Part B: Cell-Free Protein Synthesis | Cell-Free Reagents

  • E. coli Lysate BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) It contains the cellular machinery required for gene expression, including ribosomes, tRNAs, translation factors, metabolic enzymes, and aminoacyl-tRNA synthetases. It also provides T7 RNA polymerase, which transcribes DNA templates carrying a T7 promoter into mRNA.
  • Salts/Buffer
  1. Potassium Glutamate: maintains intracellular-like ionic strength and supports ribosome stability and enzyme activity.
  2. HEPES-KOH pH 7.5: buffers the reaction, keeping the pH stable for optimal transcription and translation.
  3. Magnesium Glutamate: provides Mg²⁺, an essential cofactor for ribosomes, RNA polymerase, ATP-utilizing enzymes, and nucleotide interactions.
  4. Potassium phosphate monobasic and dibasic: form a phosphate buffer pair that stabilizes pH and supplies phosphate for nucleotide phosphorylation and energy metabolism.
  • Energy / Nucleotide System
  1. Ribose: serves as the sugar backbone precursor for nucleotide biosynthesis via salvage pathways.
  2. Glucose: provides a slow, sustained energy source through glycolysis, enabling long-duration protein synthesis.
  3. AMP, CMP, GMP, and UMP: they are nucleoside monophosphates that are enzymatically converted into their triphosphate forms (ATP, CTP, GTP, and UTP), which are required for RNA synthesis and energy transfer.
  4. Guanine: acts as a precursor for GMP production through the purine salvage pathway, replenishing guanine nucleotide pools.
  • Translation Mix (Amino Acids)
  1. 17 Amino Acid Mix supplies the majority of amino acids required for protein synthesis.
  2. Tyrosine is added separately because it has limited solubility in concentrated amino acid mixtures.
  3. Cysteine is also added separately because it is chemically unstable and readily oxidizes during storage, it also forms disulfure bounds.
  • Additives
  1. Nicotinamide: supports regeneration of NAD⁺, which is essential for redox balance and sustained metabolic activity in the lysate.
  • Backfill
  1. Nuclease Free Water: adjusts the final reaction volume while protecting DNA and RNA from nuclease contamination.
  • 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 system uses PEP as a high-energy phosphate donor and provides all four nucleotides directly as NTPs, allowing rapid transcription and high initial protein production. In contrast, the 20-hour system relies on glucose for gradual ATP regeneration and supplies nucleotides as monophosphates (NMPs) plus ribose, which the lysate converts into NTPs over time, making the reaction more sustainable and cost-effective for long incubations. A second key difference is that the long-duration mix simplifies the additive package: it removes components like spermidine, cAMP, folinic acid, and NAD, replacing them with nicotinamide to support prolonged metabolic activity while reducing complexity and cost.

  • Bonus question: How can transcription occur if GMP is not included but Guanine is? Although GMP is not supplied directly, the lysate contains enzymes of the purine salvage pathway that convert guanine into GMP via guanine phosphoribosyltransferase. GMP is then phosphorylated to GDP and finally GTP, which is the actual substrate required by T7 RNA polymerase for RNA synthesis.

Part C: 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 is engineered for exceptionally robust folding, allowing it to fold efficiently even under suboptimal conditions or when fused to other proteins. Its rapid maturation and high folding efficiency make it one of the brightest and most reliable reporters in cell-free systems.

mRFP1 matures more slowly than most green fluorescent proteins, so fluorescence appears later during incubation. It also has lower intrinsic brightness, which can reduce signal intensity in short or resource-limited cell-free reactions.

mKO2 has relatively fast maturation for an orange fluorescent protein, making it well suited for time-sensitive expression assays. However, its fluorescence is somewhat sensitive to acidic conditions, so pH drift during long incubations can reduce signal.

mTurquoise2 has an exceptionally high quantum yield, making it one of the brightest cyan fluorescent proteins available. However, like many CFPs, its chromophore formation and fluorescence are highly dependent on proper folding and sufficient oxygen availability.

mScarlet-I combines very high brightness with improved maturation kinetics compared with older red fluorescent proteins. Its efficient folding and rapid chromophore formation make it particularly effective for long-term cell-free fluorescence production.

Electra2 is designed for enhanced brightness and/or unique spectral properties, but like many engineered fluorescent proteins, its performance can be sensitive to folding conditions and redox balance. Chromophore maturation is also oxygen-dependent, which can become limiting in dense or long-duration cell-free reactions.

  • 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 mScarlet-I, increasing the concentration of nicotinamide and slightly optimizing magnesium glutamate could improve long-term fluorescence output. Nicotinamide would help sustain NAD⁺ regeneration and metabolic activity over extended incubation, while optimized magnesium would enhance ribosome function and protein synthesis without causing aggregation. Hypothesis: Increasing nicotinamide and fine-tuning magnesium glutamate will improve mScarlet-I folding, maturation, and sustained expression, resulting in higher total red fluorescence after 36 hours. Alternatively, for oxygen-dependent proteins such as mTurquoise2 or Electra2, reducing reaction volume or increasing the air-liquid interface could enhance oxygen diffusion, accelerating chromophore maturation and increasing final fluorescence intensity.