Week 11 HW: Bioproduction & Cloud Labs

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

Unfortunately, I was not able to contribute to the canvas that week as I had no signal. The idea of people from all over the world collaborating on a single piece, everyone bringing their own little piece of creativity into something unified through synthetic biology, is such a cool concept.

What I really liked was how the project showcased everyone’s creativity in such a tangible way. I also loved reading the comments where people were playfully “fighting” over pixels and trying not to get their work erased. That kind of interaction made it feel like a real community experience rather than just an assignment.

For next year, it would be great if the editing window opened earlier or lasted longer, maybe two full weeks instead of just three days. I really wanted to participate. It would also be cool if there was some kind of live view or timelapse so you could watch the artwork evolve in real time, seeing how the whole thing came together pixel by pixel would be a good addition to the experience.

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

1. Component Roles in the NMP-Ribose Cell-Free Reaction

E. coli Lysate

  • BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): The lysate provides the core cellular machinery required for transcription and translation, including ribosomes, tRNAs, translation factors, and metabolic enzymes. The inclusion of T7 RNA Polymerase enables efficient transcription of genes placed under a T7 promoter.

Salts/Buffer

  • Potassium Glutamate: Serves as the primary monovalent salt in the reaction, maintaining ionic strength and stabilizing ribosome activity and protein folding.
  • HEPES-KOH pH 7.5: Acts as a buffering agent to maintain a stable physiological pH throughout the reaction, which is critical for enzymatic activity.
  • Magnesium Glutamate: Provides magnesium ions essential for ribosome assembly, stabilization of nucleic acid structures, and as a cofactor for many enzymatic reactions.
  • Potassium Phosphate Monobasic & Dibasic (1.6:1 ratio): Together these contribute to phosphate buffering capacity and provide inorganic phosphate that supports metabolic reactions within the system.

Energy / Nucleotide System

  • Ribose: Serves as a simple sugar precursor that, together with cellular enzymes from the lysate, can be used to regenerate nucleotides via the pentose phosphate pathway, providing a sustainable energy and nucleotide source.
  • Glucose: Functions as an additional carbon and energy source, feeding into central metabolic pathways to support ATP regeneration and overall reaction sustainability.
  • AMP, CMP, UMP: These nucleoside monophosphates serve as precursors that are phosphorylated by endogenous kinases into their triphosphate forms (ATP, CTP, UTP), which are then directly used as energy currency and building blocks for RNA synthesis.
  • GMP: Similarly serves as a precursor to GTP, supporting both transcription (as a nucleotide building block) and energy-dependent translation processes — though notably listed at 0.00 µM in this formulation (see Bonus).
  • Guanine: Provides a nucleobase that can be salvaged by the cell’s purine salvage pathway enzymes present in the lysate to synthesize GMP and ultimately GTP, compensating for the absence of exogenously added GMP.

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: Supplies the standard amino acids required as substrates for ribosomal translation of the target protein, excluding those provided separately.
  • Tyrosine: Provided separately due to its low solubility at neutral pH, requiring preparation at pH 12 before addition; it is an essential amino acid for protein synthesis.
  • Cysteine: Also supplied separately as it is particularly sensitive to oxidation and must be handled carefully; it is essential for proteins requiring disulfide bonds or cysteine residues.

Additives

  • Nicotinamide: Serves as a precursor to NAD⁺, supporting redox reactions and metabolic pathways within the lysate that are necessary for sustained energy regeneration.

Backfill

  • Nuclease-Free Water: Used to bring the reaction to the final desired volume without introducing RNases or DNases that would degrade the nucleic acid components of the reaction.

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

The most fundamental difference lies in how each system supplies energy and nucleotides. The 1-hour PEP-NTP mix provides energy directly through phosphoenolpyruvate (PEP) as a high-energy phosphate donor and supplies pre-formed NTPs (ATP, GTP, CTP, UTP), enabling rapid transcription and translation but depleting quickly. In contrast, the 20-hour NMP-Ribose-Glucose mix uses a more metabolically regenerative strategy, supplying nucleoside monophosphates and simple sugars (ribose and glucose) that are processed by enzymes in the lysate to continuously regenerate NTPs, thereby sustaining the reaction over a much longer period. Additionally, the 20-hour formulation includes fewer additives (e.g., no spermidine, DMSO, cAMP, NAD, or folinic acid) but adds nicotinamide to support NAD⁺ regeneration, reflecting an overall shift toward a simpler, more sustainable, and cost-effective design.

3. Bonus: How Can Transcription Occur if GMP Is Listed at 0.00 µM?

Transcription can still occur because guanine is included in the reaction and can be converted to GMP, then to GTP, through the purine salvage pathway. Enzymes present in the E. coli lysate, such as hypoxanthine-guanine phosphoribosyltransferase (HGPRT), catalyze the transfer of a ribose-5-phosphate group from phosphoribosyl pyrophosphate (PRPP) onto guanine to generate GMP directly. This GMP is then phosphorylated to GDP and GTP by nucleoside monophosphate and diphosphate kinases also present in the lysate, making exogenous GMP unnecessary as long as the salvage pathway remains active.

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

1. Biophysical/Functional Properties of Each Fluorescent Protein

i. sfGFP (Superfolder GFP)

sfGFP was specifically engineered to fold more robustly and faster than standard reporter GFP, and its maturation process involves folding of the β-barrel, torsional rearrangements, cyclization, and oxidation of the chromophore. This exceptionally fast and robust folding makes sfGFP one of the most reliable reporters in cell-free systems, where molecular chaperones are limited and proteins must fold without cellular assistance. sfGFP demonstrated a 3.5-fold faster initial folding rate compared to its predecessor and tolerated challenging fusion partners, making it highly suitable for cell-free expression contexts.

ii. mRFP1

mRFP1 is reported to be a somewhat slowly-maturing monomer, and although it has a lower extinction coefficient, quantum yield, and photostability than DsRed, it matures more than 10 times faster than its tetrameric predecessor. In a cell-free context, the slow maturation relative to sfGFP is a relevant limitation, as the oxidation step required for chromophore formation depends on available dissolved oxygen in the reaction, meaning fluorescence signal readout may be delayed and underrepresent total protein yield, particularly in longer incubation formats.

iii. mKO2 (monomeric Kusabira Orange 2)

mKO2 shows a strong dependence on oxygen tension during the maturation process, and its fluorescence recovery kinetics after reoxygenation are notably slower than those of green fluorescent proteins. This heightened oxygen dependence is particularly relevant in cell-free systems, where oxygen availability can be limited in sealed or anaerobic reaction conditions, meaning mKO2 may yield a significantly reduced fluorescence signal even if the protein is being actively translated.

iv. mTurquoise2

mTurquoise2 is reported to be a rapidly-maturing monomer with very low acid sensitivity. Its exceptionally high quantum yield, improved photostability, and faster maturation relative to its predecessor mTurquoise make it well-suited for cell-free systems, where the pH of the reaction mixture can drift over long incubation periods — the low acid sensitivity ensures that fluorescence readout remains stable and reliable throughout the 20- or 36-hour incubation windows.

v. mScarlet-I

The single amino acid substitution T74I found in mScarlet-I results in a marked maturation acceleration in cells, but at the cost of a moderate decrease in fluorescence quantum yield (0.54) and fluorescence lifetime (3.1 ns), although both values are still higher than those of all previously engineered bright monomeric RFPs. In a cell-free context, this trade-off is generally favorable, faster maturation means a greater fraction of translated protein becomes fluorescent within the incubation window, making mScarlet-I a better real-time reporter than the brighter but slower-maturing mScarlet.

vi. Electra2

Electra2 is a monomeric blue fluorescent protein engineered with optimized intracellular brightness, developed through hierarchical screening in both bacterial and mammalian cells. A relevant functional property for cell-free systems is that, like all blue fluorescent proteins, Electra2 requires molecular oxygen for chromophore maturation and is derived from a GFP-like scaffold — blue FPs as a class are characterized by lower brightness compared to green, yellow, and red counterparts, which means that Electra2’s signal-to-noise ratio in a cell-free readout will be inherently lower and may require longer incubation times or higher DNA template concentrations to produce a detectable fluorescent signal.

2. Hypothesis for Mastermix Optimization

Protein: mKO2 As described above, mKO2 exhibits a strong dependence on oxygen availability during the final oxidation step of chromophore maturation. In a sealed cell-free reaction run over 36 hours, dissolved oxygen in the mastermix is progressively consumed by metabolic activity in the lysate, which is expected to severely limit mKO2 maturation over time, even if transcription and translation remain productive.

Hypothesis: Supplementing the 36-hour cell-free mastermix with a controlled oxygen-releasing compound, such as a dilute concentration of hydrogen peroxide (H₂O₂) or a solid oxygen-releasing material, or alternatively conducting the reaction in a gas-permeable vessel with atmospheric oxygen exchange, would increase the available dissolved oxygen concentration throughout the incubation period. This is expected to accelerate and sustain chromophore oxidation in mKO2, resulting in a greater fraction of translated protein reaching the fully mature, fluorescent state and therefore producing a higher cumulative fluorescence signal at the 36-hour endpoint compared to a standard sealed reaction.

3. Composition

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Part D: Build-A-Cloud-Lab | (optional) Bonus Assignment