Bioproduction & Cloud Labs

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

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

E.Coli Lysate:

It is acted as the catalytic core of the reaction; it provides the ribosomes for translation and T7 RNA Polymerase for high-level transcription.

Salts and Buffers:

  • Potassium Glutamate: It provides necessary ionic strength and potassium ions for protein folding without inhibiting DNA-protein interactions like chloride salts might.
  • HEPES-KOH pH 7.5: It is pH indicator.Also it prevents reaction from becoming too acidic due to metabolic byproducts.
  • Magnesium Glutamate: It supplies magnesium ions required for stabilizing ribosome assembly and acting as a cofactor for RNA polymerase.
  • Potassium Phosphate (Monobasic- Dibasic): It provides secondary buffering and source of inorganic phosphate needed to regenerate ATP.

Energy and Nucleotide System:

  • Ribosome
  • Guanine: It serves as specific region for nitrogenous base that can be salvaged by lysate enzymes to create GTP for transcription when direct GMP levels are low.

Translation Mix (Amino Acids):

they are provide fundamental building blocks for synthesizing the polypeptide chain.

Additives:

  • Nicotinamide: Stabilizes metabolis cofactors like NAD+, preventing their degradation and ensuring energy generating pathways reman active.

Backfill

  • Nuclease Free Water: Acts as solvent for reaction, ensuring no DNA/RNA-degrading enzymes interfere with genetic templates.
  1. 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.

The 1-hour optimized PEP-NTP master mix utilizes a high-energy phosphate donor (PEP) for rapid, burst-like ATP production, which is ideal for short, high-speed reactions but leads to quick energy depletion and pH drops. In contrast, the 20-hour NMP-Ribose-Glucose master mix relies on a slower, more sustainable metabolic recycling of nucleotides and sugars, allowing the reaction to maintain productivity and pH stability for nearly a day, resulting in a much higher total protein yield.

  1. Bonus question: How can transcription occur if GMP is not included but Guanine is? Transcription can still occur through nucleotide salvage pathways present in the E. coli lysate. Enzymes such as phosphoribosyltransferases can take the Guanine base and chemically link it to a ribose-phosphate molecule to form GMP, which is then further phosphorylated by the energy system into GTP, the required substrate for RNA polymerase.

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)

  • sfGFP (Superfolder GFP): Known for its extremely fast folding and high stability; it is the “gold standard” for cell-free systems because it folds correctly even when fusion tags are attached.

  • mRFP1 (monomeric Red Fluorescent Protein): While a classic red marker, it is known for having a relatively slow maturation time and lower brightness compared to newer red variants like mScarlet.

  • mKO2 (monomeric Kusabira Orange 2): A bright orange protein that is highly pH-stable but can be sensitive to oxygen levels for proper chromophore maturation.

  • mTurquoise2: An exceptionally bright cyan protein with a high quantum yield and high photostability, though it requires specific filter sets for accurate readout.

  • mScarlet-I: One of the brightest red fluorescent proteins available; its main advantage in cell-free systems is its high brightness and faster maturation compared to mRFP1.

  • Electra2: A newer generation protein designed for high-level expression; it is often optimized for minimal metabolic burden on the cell-free machinery.

  1. 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.

  • Protein: mScarlet-I

  • Reagent Adjustment: Increase the concentration of the 17 amino acid mix and magnesium glutamate.

  • Expected Effect: Since mScarlet-I is a highly bright and “demanding” protein, increasing the amino acid supply will ensure that translation doesn’t stall during the long 36-hour incubation. Boosting Magnesium will further stabilize the ribosomes for this extended run, ideally resulting in a steeper fluorescence curve and a higher final signal-to-noise ratio.

  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.

For my custom 2 $\mu$L supplement in the 20 $\mu$L reaction, I plan to focus on molecular crowding agents (like PEG) or additional energy boosters (like extra Glucose) to see if we can sustain the reaction beyond the typical 20-hour window.