Week 11 HW: Bioproduction & Cloud Labs

Part A: The 1,536 Pixel Artwork Canvas

Contribution I designed a small section of the artwork using a few pixels to again create the BTS pop band logo, cos i am obsessed. Might not look like it, as the pixels were already too full.

I liked how different ideas were combined into one large artwork. For next time, Divided sections for nodes could make collective teamwork visible.

Part B: Cell-Free Protein Synthesis

E. coli Lysate-BL21 (DE3) Star Lysate Provides ribosomes, enzymes, and T7 RNA polymerase needed for transcription and translation.

Salts/Buffer Maintain pH, ion balance, and ribosome stability during protein synthesis.

Energy / Nucleotide System Provides energy and nucleotide precursors required for RNA production and ATP regeneration.

Translation Mix (Amino Acids) Supplies amino acids needed to build proteins.

Additives: Nicotinamide Helps protect nucleic acids and supports redox balance.

Backfill: Nuclease-Free Water Adjusts reaction volume without introducing contaminants.

1-Hour PEP-NTP vs 20-Hour NMP-Ribose-Glucose Mix The PEP-NTP mix gives fast protein production but runs out of energy quickly. The NMP-Ribose-Glucose mix regenerates energy slowly, allowing protein expression for many hours.

Bonus Question Free guanine is converted into GMP by enzymes in the lysate. GMP is then converted into GTP for transcription.

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

sfGFP Folds quickly and gives fast fluorescence, making it reliable in cell-free systems.

mRFP1 Matures slowly, so fluorescence appears later but remains stable.

mKO2 Produces strong orange fluorescence but is sensitive to acidic conditions.

mTurquoise2 Very bright cyan protein but requires correct folding conditions.

mScarlet_I Bright red fluorescent protein that needs oxygen for maturation.

Electra2 Stable blue fluorescent protein but may aggregate under some salt conditions.

Hypothesis Using extra glucose and magnesium with better oxygen exposure will improve mScarlet_I fluorescence over 36 hours by supporting energy production and chromophore maturation.

Alternative Hypothesis Adding nicotinamide and cysteine may improve mTurquoise2 folding and produce stronger cyan fluorescence.

Master Mix Design The mix includes glucose for energy, magnesium for translation, cysteine and nicotinamide for stability, and buffers to maintain pH and ionic balance.