Week 11 HW: Bioproduction

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

Reflection on the Community Bioart Project

R/: I contributed one pixel in the upper-right area of the collaborative canvas. Although it was a small contribution, it became part of a collective bioart piece created by students from around the world.

What I liked most about this project was the idea of global collaboration. Watching how individual contributions, even something as small as a single pixel, combined to form a unified artwork was a beautiful reflection of how collective science and art can work together.

For next year, I think the project could be improved by adding a live update feature so contributors could watch the canvas evolve in real time. It would also be interesting to include a small profile or identifier showing who contributed each pixel, making the human connection behind each contribution more visible.

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

1. Roles of Each Component in the Cell-Free Reaction

E. coli Lysate

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

R/: The BL21 (DE3) Star lysate provides all the cellular machinery required for transcription and translation, including ribosomes, tRNAs, metabolic enzymes, and translation factors. It also contains T7 RNA Polymerase, which specifically transcribes genes under the control of a T7 promoter.

Salts and Buffer Components

Potassium Glutamate

R/: Potassium glutamate maintains ionic strength and stabilizes the transcription and translation machinery, mimicking the intracellular ionic environment of E. coli.

HEPES-KOH pH 7.5

R/: HEPES-KOH acts as a buffer that maintains the reaction pH near 7.5, which is optimal for enzymatic activity during transcription and translation.

Magnesium Glutamate

R/: Magnesium glutamate provides Mg²⁺ ions, which are essential cofactors for ribosomes, RNA polymerase, and many enzymes involved in protein synthesis.

Potassium Phosphate Monobasic/Dibasic

R/: These phosphate salts help stabilize pH and provide phosphate ions required for energy metabolism and nucleotide synthesis.

Energy / Nucleotide System

Ribose

R/: Ribose is a pentose sugar that serves as a precursor for nucleotide biosynthesis, helping regenerate NTPs required for transcription.

Glucose

R/: Glucose acts as a carbon and energy source that fuels metabolic pathways involved in ATP regeneration during translation.

AMP, CMP, GMP, UMP

R/: These nucleoside monophosphates serve as precursors for RNA synthesis and are phosphorylated into their triphosphate forms (NTPs) used during transcription.

Guanine

R/: Guanine can be salvaged and converted into GMP/GTP, providing an additional source of guanine nucleotides for RNA synthesis.

Translation Mix

17 Amino Acid Mix

R/: The amino acid mix provides 17 of the 20 standard amino acids required as substrates for protein synthesis.

Tyrosine

R/: Tyrosine is added separately because it has low solubility and must be prepared independently to ensure sufficient concentration in the reaction.

Cysteine

R/: Cysteine is added separately because it is prone to oxidation and must remain in its reduced form for efficient protein synthesis.

Additives

Nicotinamide

R/: Nicotinamide acts as a precursor for NAD⁺, an essential cofactor involved in metabolic reactions that regenerate ATP during the cell-free reaction.

Backfill

Nuclease-Free Water

R/: Nuclease-free water is used to bring the reaction to its final volume without introducing RNases or DNases that could degrade RNA or DNA templates.

2. Differences Between the 1-Hour Optimized PEP-NTP Master Mix and the 20-Hour NMP-Ribose-Glucose Master Mix

R/: The 1-hour optimized PEP-NTP master mix uses phosphoenolpyruvate (PEP) as the primary energy source and directly supplies nucleoside triphosphates (NTPs), allowing rapid and efficient protein production during short reactions.

In contrast, the 20-hour NMP-Ribose-Glucose master mix uses nucleoside monophosphates (NMPs) together with ribose and glucose, relying on endogenous metabolic enzymes in the lysate to regenerate NTPs gradually over time. This system is more cost-effective and better suited for long-duration protein expression.

3. Bonus Question: How Can Transcription Occur if GMP Is Not Included but Guanine Is?

R/: Although GMP is not directly included, transcription can still occur because guanine can be salvaged by enzymes present in the E. coli lysate. Enzymes such as hypoxanthine-guanine phosphoribosyltransferase (HGPRT) convert guanine into GMP using phosphoribosyl pyrophosphate (PRPP), and GMP is subsequently phosphorylated into GDP and GTP, which are required for RNA synthesis by RNA polymerase.

Part C: Planning the Global Experiment | Cell-Free Master Mix Design

1. Biophysical or Functional Properties of the Fluorescent Proteins

sfGFP

R/: sfGFP (Superfolder GFP) has exceptionally fast and robust folding kinetics, allowing maturation in under 10 minutes at 37°C. This makes it highly suitable for cell-free systems where reaction time is limited. However, like all GFP derivatives, it requires molecular oxygen for chromophore maturation.

mRFP1

R/: mRFP1 has incomplete chromophore maturation, meaning that a fraction of the synthesized protein never becomes fluorescent. It also has relatively low photostability and quantum yield, which can reduce fluorescence intensity in cell-free systems.

mKO2

R/: mKO2 is moderately sensitive to acidic conditions, meaning that slight decreases in pH during long incubations can reduce fluorescence output. Proper buffering is therefore important for maintaining signal stability.

mTurquoise2

R/: mTurquoise2 undergoes slow two-step chromophore maturation, making it one of the slowest-maturing cyan fluorescent proteins. In short cell-free reactions, fluorescence may underestimate the total amount of synthesized protein.

mScarlet-I

R/: mScarlet-I is known for robust folding and efficient maturation, even under oxidizing conditions. This makes it a reliable reporter protein in cell-free systems with varying redox environments.

Electra2

R/: Electra2 is a bright blue fluorescent protein with improved fluorescence compared to many other blue fluorescent proteins. However, like other β-barrel fluorescent proteins, it requires molecular oxygen for chromophore maturation, making oxygen availability important for long incubations.

2. Hypothesis for Improving Fluorescence Over a 36-Hour Incubation

R/: I hypothesize that increasing the concentrations of glucose and ribose in the cell-free master mix would improve the fluorescence output of mTurquoise2 during a 36-hour incubation.

Because mTurquoise2 undergoes slow two-step chromophore maturation, it requires extended reaction times to achieve maximum fluorescence. By increasing glucose and ribose concentrations, ATP regeneration could be sustained for longer periods, maintaining active translation and allowing a larger fraction of synthesized mTurquoise2 proteins to complete chromophore maturation. As a result, higher fluorescence intensity would be expected at later time points (18–36 hours) compared to the standard master mix composition.