Week 11 HW: Bioproduction and Cloud Labs

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

I missed the date to contribute :c Will try my best to become a TA next year!

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

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.

  1. Component Roles

Component Category and their role in the Reaction:

E. coli Lysate : Provides the essential molecular “machinery,” including ribosomes and translation factors; the Star mutation in RNase E prevents mRNA degradation to increase yields, while the DE3 lysogen provides T7 RNA Polymerase for high-level transcription.

Salts / Buffers: HEPES-KOH maintains a stable pH (~7.5) necessary for enzyme function; Potassium and Magnesium salts act as essential cofactors that stabilize the ribosome structure and facilitate the assembly of the translation complex.

Energy / Nucleotide System: Ribose and Glucose serve as metabolic precursors to regenerate energy; the NMPs (AMP, CMP, GMP, UMP) are phosphorylated into the high-energy triphosphates (ATPs, etc.) required for fueling both transcription and translation.

Translation Mix: Provides the 20 building blocks (amino acids) required to assemble the protein chain; Tyrosine and Cysteine are often added separately or in excess because they have lower solubility or are more easily degraded in extract.

Additives: Nicotinamide (part of NAD) acts as a crucial co-factor for redox reactions, helping maintain metabolic flow and preventing the depletion of energy substrates during the reaction.

Backfill: Nuclease-Free Water is used to reach the final reaction volume without introducing enzymes that would destroy the DNA template or mRNA.

  1. Master Mix Comparisons

The 1-hour PEP-NTP mix is designed for speed and “burst” production, utilizing the high-energy substrate Phosphoenolpyruvate (PEP) and pre-supplied nucleotide triphosphates to reach peak yields rapidly before the energy is exhausted. In contrast, the 20-hour NMP-Ribose-Glucose mix is optimized for longevity; it relies on slower internal metabolic pathways to regenerate energy and nucleotides from cheaper, more stable precursors, resulting in a lower initial expression rate but sustained production over a much longer period.

  1. Bonus: Transcription without GMP?

Transcription can occur even if GMP (Guanosine Monophosphate) is missing because the system contains Guanine and the necessary enzymes within the E. coli lysate to perform salvage pathways. Specifically, the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) can convert the Guanine base into GMP by attaching a ribose-phosphate group. Once GMP is formed, it is further phosphorylated by kinases in the lysate into GTP, which is the actual substrate used by RNA polymerase to build the RNA chain.

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)

  1. Biophysical & Functional Properties

    sfGFP (superfolder GFP): This protein is engineered for extremely fast folding and high thermodynamic stability, which allows it to reach peak fluorescence quickly in cell-free systems even when ribosomes are limited.

    mRFP1 (monomeric Red Fluorescent Protein): This protein has a relatively slow maturation time (the time it takes for the internal chromophore to chemically “bond” after folding), which can lead to a significant lag between protein production and visible color.

    mKO2 (monomeric Kusabira Orange 2): It is highly sensitive to pH levels; if the cell-free reaction becomes too acidic due to metabolic byproduct accumulation (like lactic acid), the orange fluorescence will dim significantly.

    mTurquoise2: This variant has a very high quantum yield (brightness) but requires specific “gate-keeping” residues to stay folded; it is particularly sensitive to the concentration of molecular chaperones present in the lysate.

    mScarlet-I: While it is the brightest monomeric red protein, its chromophore formation is highly oxygen-dependent, meaning it may underperform in thick hydrogels or deep “wells” where oxygen diffusion is restricted.

    Electra2: Designed for rapid “photo-activation” or high-speed maturation, its primary constraint in cell-free systems is its tendency to aggregate if the translation rate is too high, requiring a balanced synthesis speed to stay soluble.

  2. Optimization Hypothesis

Protein: mScarlet-I

Reagents: Potassium Phosphate (Buffer) and Nicotinamide (Additives)

Hypothesis: By increasing the concentration of Potassium Phosphate and adding a secondary redox-active Nicotinamide/NAD+ cocktail, we can stabilize the pH and maintain metabolic flow for a full 36-hour run.

Reasoning: In long-term (36-hour) incubations, the primary threat to mScarlet-I is the “stalling” of chromophore maturation. This maturation is an oxidative process. As the reaction progresses, the environment often becomes more acidic (reducing brightness) and oxygen is depleted.

  • Increasing the Phosphate buffer prevents the pH drop that would otherwise quench the red signal.

  • Optimizing the Nicotinamide levels helps maintain the metabolic “engine” that handles oxygen-consuming byproducts, ensuring that available dissolved oxygen is directed toward the mScarlet-I chromophore formation rather than just being sucked up by secondary metabolic stress.

  • Expected Effect: This should result in a deeper, more saturated red hue that continues to intensify throughout the 36-hour window rather than plateauing at hour 8.