Week 11 HW: Bioproduction and Cloud Labs

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

I was not able to complete the artwork since I never received the email :c.

2. 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.

E. coli Lysate

BL21 (DE3) Star Lysate (includes T7 RNA Polymerase)

This lysate provides the cellular machinery required for transcription and translation, including ribosomes, enzymes, tRNAs, and metabolic components. The T7 RNA polymerase specifically drives the transcription of genes under a T7 promoter, enabling protein synthesis in the cell-free system.

Salts/Buffer

Potassium Glutamate: Helps maintain ionic strength and mimics the intracellular environment of E. coli. It supports enzyme activity and improves protein synthesis efficiency.

HEPES-KOH pH 7.5: Acts as a buffering agent to maintain a stable pH during the reaction. Proper pH is essential for optimal enzyme and ribosome activity.

Magnesium Glutamate: Stabilize ribosomes, nucleic acids, and enzymatic reactions involved in protein synthesis as they are essential cofactors for transcription and translation processes.

Potassium phosphate monobasic: Contributes to phosphate buffering capacity and helps maintain the chemical balance of the reaction mixture.

Potassium phosphate dibasic: Works together with the monobasic form to stabilize pH and provide phosphate ions necessary for cellular reactions.

Energy / Nucleotide System

Ribose: Serves as a carbon source and can contribute to energy regeneration pathways during the reaction.

Glucose: Provides metabolic energy that supports ATP regeneration and prolongs protein synthesis activity in the cell-free system.

AMP: A nucleotide precursor involved in ATP regeneration and nucleic acid metabolism during transcription.

CMP: Provides cytidine nucleotides required for RNA synthesis during transcription.

GMP: Supplies guanine nucleotides necessary for mRNA production.

UMP: Provides uridine nucleotides used during RNA synthesis.

Guanine: Supports nucleotide biosynthesis and recycling pathways that help sustain transcription.

17 Amino Acid Mix

This mixture provides most of the amino acids required for protein synthesis. Ribosomes use these amino acids to assemble the target protein.

Tyrosine: It is added separately to ensure sufficient availability during translation, since it may be unstable or consumed rapidly. Cysteine: It is supplied separately because it is chemically reactive and prone to oxidation, which can reduce its stability in the mixture.

Additives

Nicotinamide: Nicotinamide functions as a precursor for NAD-related cofactors that support metabolic and enzymatic reactions in the cell-free system.

Backfill

Nuclease Free Water: Nuclease free water is used to adjust the final reaction volume while preventing degradation of nucleic acids by nucleases.

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 optimized PEP-NTP master mix uses phosphoenolpyruvate (PEP) and directly supplied NTPs as an immediate energy and transcription source, allowing rapid protein production in a short incubation time. In contrast, the 20-hour NMP-Ribose-Glucose master mix relies on ribose, glucose, and nucleotide monophosphates (NMPs), which are metabolically converted into usable energy and nucleotides over time through endogenous cellular enzymes.

Additionally, the 20-hour system is designed to be more sustainable and cost-effective for long incubations, while the 1-hour system prioritizes fast and high-yield protein expression.

3. 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)

Table 1: Protein properties

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.

Increasing the concentration of ribose and glucose in the 20-hour NMP-Ribose-Glucose cell-free mastermix could improve the fluorescence output of mScarlet-I during a 36-hour incubation

Because mScarlet-I depends on efficient folding and chromophore maturation over longer expression periods, providing additional long-term energy substrates may sustain ATP regeneration and protein synthesis, resulting in brighter and more stable fluorescence over time. Another possible modification would be increasing magnesium glutamate concentration slightly to improve ribosome activity and translation efficiency, which could further enhance mScarlet-I expression. Tiny chemical soup optimization rituals.

Bonus question: How can transcription occur if GMP is not included but Guanine is?

Transcription can still occur because the system may use guanine as a precursor to synthesize GMP and eventually GTP through endogenous metabolic enzymes present in the cell extract. In the NMP-Ribose-Glucose system, the cell-free lysate retains parts of cellular nucleotide salvage pathways, allowing guanine to be converted into usable nucleotide forms needed for RNA synthesis over long incubations.