Week 11 HW: BIOPRODUCTION & CLOUD LABS

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

I helped with my community’s HTGAA: 1536 project, on the top right part, by adding a tongue — or what looks to me like a tongue — to the green head. I liked how different ideas came together to create something like art, and I feel that really represents what it’s like to work as a team in any discipline — especially in the sciences, where different worlds must come together for a shared goal.

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

E. coli Lysate

Provides the endogenous cellular machinery (ribosomes, tRNA, aminoacyl-tRNA synthetases, and metabolic enzymes) necessary for transcription and translation, with the BL21 (DE3) Star strain specifically containing T7 RNA polymerase for high-yield gene expression.

Salts/Buffer

  • Potassium Glutamate: Maintains ionic strength and provides a physiological potassium environment that optimizes ribosome function and protein folding without inhibiting enzymatic activity.

  • HEPES-KOH pH 7.5: Acts as a buffering agent to maintain the reaction at a stable pH 7.5, ensuring optimal activity for all enzymes in the cell-free system.

  • Magnesium Glutamate: Serves as an essential cofactor for ribosome structure and RNA polymerase activity, where free magnesium concentration critically influences transcription and translation fidelity.

  • Potassium phosphate monobasic & dibasic: Work together as a phosphate buffer system to maintain pH stability and provide inorganic phosphate needed for ATP regeneration.

Energy / Nucleotide System

  • Ribose: Supplies the pentose sugar backbone for de novo nucleotide synthesis via the pentose phosphate pathway, allowing sustained energy and nucleotide regeneration over extended incubations.

  • Glucose: Serves as the primary carbon and energy source, feeding into glycolysis and the pentose phosphate pathway to power ATP and nucleotide regeneration.

  • AMP, CMP, UMP, GMP & Guanine: Provide nucleotide monophosphate precursors and a guanine base, which are converted by endogenous E. coli enzymes into the NTPs required for transcription (ATP, CTP, UTP, GTP).

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: Supplies the building blocks for protein synthesis, omitting tyrosine and cysteine which are added separately to prevent chemical precipitation or degradation.

  • Tyrosine: An amino acid added separately at high pH (pH 12) to maintain solubility before being neutralized in the reaction mix.

  • Cysteine: A sulfur-containing amino acid added separately to prevent oxidation and disulfide bridge formation during long incubations.

Additives

  • Nicotinamide: Functions as a vitamin B3 derivative that supports NAD/NADH cofactor recycling, essential for maintaining redox balance and metabolic activity in extended reactions.

Backfill

  • Nuclease Free Water: Adjusts the master mix to the desired final volume and ensures no contaminating nucleases degrade DNA templates or RNA transcripts.

The 1-hour optimized PEP-NTP mix provides immediate, high-energy substrates (ATP, GTP, CTP, UTP, and phosphoenolpyruvate) for rapid protein production, making it suitable for short incubations. In contrast, the 20-hour NMP-Ribose-Glucose mix supplies simple precursors (AMP, CMP, UMP, guanine, ribose, and glucose) that rely on endogenous E. coli enzymes to slowly regenerate energy and nucleotides over time, enabling sustained fluorescent protein production for up to 20 hours. The long-incubation mix also replaces the synthetic energy molecule PEP with a metabolic system using glucose and ribose, and swaps several additives (e.g., removing DMSO and spermidine while adding nicotinamide) to support extended reaction stability.

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

sfGFP: Superfolder GFP has a rapid maturation time of approximately 13.6 minutes, which allows it to achieve fluorescence quickly in cell-free systems. Its superfolder property also enables proper folding even when expressed as a fusion protein, reducing the risk of aggregation and improving reliable readout

mRFP1: mRFP1 has a relatively slow maturation time of 60 minutes, which limits its initial brightness in short incubations but allows sustained signal over longer timeframes. Its low pKa of 4.5 makes it acid-tolerant, a useful property in cell-free systems where pH can drift over extended 36-hour incubations.

mKO2: mKO2 has a very slow maturation time of 108 minutes, requiring extended incubation to achieve full fluorescence, which is relevant for 36-hour artwork experiments. Its instability index of 46.21 indicates it is prone to degradation, potentially limiting signal stability over long cell-free reactions unless protease inhibitors or stabilizing reagents are added.

mTurquoise2: mTurquoise2 has an exceptionally fast maturation time of approximately 3.5 minutes, allowing it to achieve fluorescence almost immediately upon protein folding in cell-free systems. Its high photostability (t½ = 90 seconds) and stable instability index of 27.33 make it reliable for long 36-hour incubations without significant degradation.

mScarlet_I: mScarlet_I has the slowest maturation time of all six proteins at 174 minutes, requiring prolonged incubation (well beyond typical cell-free reaction times) to reach full fluorescence. However, it compensates with exceptionally high brightness (70.0) and outstanding photostability (t½ = 277 seconds), making it ideal for long-term 36-hour artwork incubations where signal persistence is valued over rapid onset.

Electra2: Electra2 exhibits exceptional photostability with a half-life of up to 1466 seconds under LED illumination, making it highly resistant to photobleaching during long-term imaging or artwork documentation. Its brightness of 61.48 and fast maturation (typical for small blue fluorescent proteins) allow rapid signal development that persists reliably over 36-hour cell-free incubations.

Protein: mScarlet_I

Biophysical property to improve: Extremely slow maturation time (174 minutes)

Reagent(s) to adjust: Increase magnesium glutamate concentration from 7.0 mM to 9.0–10.0 mM, and add 1.0 mM dithiothreitol (DTT) as a reducing agent.

Expected effect: Higher magnesium concentration accelerates the rate of chromophore cyclization and oxidation, reducing the effective maturation time of mScarlet_I by stabilizing the proper folding intermediate. The addition of DTT prevents oxidative misfolding of cysteine residues, allowing more nascent protein to reach its mature, fluorescent conformation. Together, these adjustments are expected to increase total fluorescence yield at 36 hours by 30–50% compared to the standard master mix, without compromising signal stability.