Week 11 — Bioproduction & Cloud Labs
Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork
Make a note on your HTGAA webpages including:
What you contributed to the community bioart project
I made part of the 2026 on the upper left plate
What you liked about the project, and what about this collaborative art experiment could be made better for next year.
I liked the idea of working with hundreds of people on a single collaborative project that can express the essence of all the participants. What could be improved next year would be to add more colors.
Part B: Cell-Free Protein Synthesis | Cell-Free Reagents
- E. coli Lysate
BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): Supplies a high-yield transcription system via T7 RNA polymerase, and the Star strain reduces RNase activity for increased mRNA stability.
- Salts/Buffer
Potassium Glutamate: Maintains ionic strength and mimics the intracellular environment more effectively than chloride salts, enhancing translation efficiency.
HEPES-KOH pH 7.5: Buffers the reaction mixture at an optimal pH for enzymatic activity.
Magnesium Glutamate: Serves as a necessary cofactor for ribosome function, tRNA binding, and RNA polymerase activity.
Potassium phosphate monobasic: Along with its dibasic form, helps maintain pH and supplies phosphate for energy regeneration.
Potassium phosphate dibasic: Works with the monobasic form to buffer the reaction and contribute to the phosphate pool.
- Energy / Nucleotide System
Ribose and glucose: Act as energy sources and carbon backbones to fuel metabolic pathways that regenerate ATP and GTP.
AMP, CMP, GMP y UMP: Are converted to the corresponding nucleotide triphosphates (ATP, CTP, GTP, UTP) to serve as substrates for transcription and energy metabolism.
Guanine: Can be salvaged to produce GTP, which is critical for translation initiation and elongation.
- Translation Mix (Amino Acids)
17 Amino Acid Mix: Supplies the common amino acids required for protein synthesis; typically excludes tyrosine and cysteine to allow controlled addition
Tyrosine and cysteine: Added separately to prevent chemical modification or precipitation that can occur during long-term storage of the full amino acid mix.
- Additives
Nicotinamide: Helps regenerate NAD⁺ and inhibits certain proteases, thereby improving reaction longevity and yield.
- Backfill
Nuclease Free Water: Adjusts the final volume of the reaction to achieve the desired concentrations of all components, while ensuring no contaminating nucleases degrade mRNA.
- 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 main difference is the energy and nucleotide supply strategy. The 1-hour uses pre-supplied NTPs for immediate, rapid energy regeneration, enabling fast protein synthesis. In contrast, the 20-hour uses simple precursors that are metabolically converted into NTPs over time, which supports sustained, long-term protein production.
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)
sfGFP: Robust and rapid folding even without chaperone assistance, however, its chromophore maturation still requires molecular oxygen, meaning anaerobic or oxygen-depleted CFPS reactions will show reduced fluorescence despite proper translation. sfGFP’s high resistance to aggregation makes it ideal for CFPS, but its sensitivity to acidic pH (below ~6.5) can quench fluorescence, requiring careful buffer maintenance during extended incubations
mRFP1: Slow maturation time due to its obligate requirement for chromophore oxidation and dehydration, which limits its utility in short reactions. Exhibits some sensitivity to oligomerization at high concentrations, lacking membrane compartments, this can lead to solubility issues or altered spectral properties compared to monomeric red FPs.
mKO2: Rapid maturation rate due to its efficient chromophore formation. Like all GFP-derived fluorescent proteins, mKO2 requires molecular oxygen for chromophore oxidation, meaning that oxygen depletion in extended cell-free reactions can limit final fluorescence yield despite active protein synthesis.
mTurquoise2: High quantum yield and efficient maturation, but its chromophore is sensitive to acidic pH (pKa ~5.1), meaning that any drop in pH during extended cell-free reactions can quench fluorescence readout. Additionally, it requires proper oxidation of its chromophore, making it dependent on adequate oxygen levels in the reaction mix for full fluorescent signal development.
mScarlet_I: Accelerated maturation, which is beneficial for the systems because it allows rapid fluorescence development within short reaction times. Its red chromophore requires precise oxidative maturation conditions, and any oxygen limitation or redox imbalance in the lysate can reduce final fluorescence yield. but, it exhibits excellent monomeric behavior and pH stability, making it less sensitive to mild acidification.
Electra2: High photostability, but its chromophore maturation is relatively slow and oxygen-dependent, requiring sufficient dissolved oxygen in the reaction for complete fluorescent signal development. It has lower quantum yields and are more prone to acid sensitivity, meaning that pH drops during extended incubations
- 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.
Limitation: Its chromophore maturation requires molecular oxygen, and extended 36-hour incubations lead to oxygen depletion and pH acidification, which can quench fluorescence even if the protein remains folded.
Adjust:
Long-term ATP regeneration and buffer against acidification:
Glucose increase from 6.9 mM to 15–20 mM Ribose increase from 77.4 mM to 100 mM HEPES-KOH pH 7.5 increase from 45 mM to 80 mM
Protect the oxidizing chromophore from peroxide damage:
Catalase add 100 U/mL
‘‘This combined adjustment is expected to yield stable sfGFP fluorescence for up to 36 hours, with at 1.5–2× higher endpoint signal compared to the standard 20-hour master mix, by preventing both energy collapse and oxidative stress during prolonged incubation’’
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