Week 11 HW: Bioproduction and Cloud Labs

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

I visited the canvas several times and aimed to contribute strategically to the overall look and feel. I added a yellow “MIT” at some point and contributed around 200 pixels overall, ranking approximately 10th on the contributions list last I checked.

I enjoyed the collaborative aspect and that we could all participate together independently of node and location.

One improvement could be introducing a “live hour” (or even just 5 minutes during homework review) where everyone gathers on Zoom to paint together. This might foster more online connection, increase engagement from some students, and spark casual conversation within HTGAA about the project. I also think large automated scripts should be discouraged as if pixels are placed randomly or too particular/specific to something, it defeats the collaborative element and reduces the chance of organic group design outcomes emerging naturally.

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

E. coli BL21 Star lysate with T7 RNA polymerase: Contains the ribosomes and enzymes needed to read DNA and build proteins. The T7 polymerase specifically recognizes and reads the T7 promoter on a DNA template.

Salts/Buffer (potassium glutamate, HEPES, magnesium): Maintains the correct pH and ionic environment so the cellular machinery can function properly.

Energy/Nucleotide system (ribose, glucose, NMPs): Provides the energy molecules and building blocks needed to synthesize RNA and power the protein synthesis reactions.

Translation mix (amino acids): Supplies the 20 amino acids that the ribosome links together in the correct order to build the protein chain.

Tyrosine specifically: Acts as a key component of the chromophore in fluorescent proteins, enabling them to fluoresce.

Cysteine:

Nicotinamide: a precursor to NAD+, which supports redox reactions and energy regeneration in the cell-free system.

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

mTurquoise2: A 36 hour (48-hour at hw review?) cell-free reaction time for this experiment means that mTurquoise2, which has a slower maturation time, is not a major constraint since there is sufficient incubation time for full chromophore maturation.

sfGFP: sfGFP has robust folding capability, which allows it to fold correctly without cellular chaperones, resulting in faster and more efficient fluorescent protein production in cell-free systems.

Electra2: Electra2’s performance in bacterial cell-free systems may be unpredictable because it was engineered and optimized for mammalian cells, not for E. coli expression environments.

mScarlet-I: mScarlet-I reaches peak fluorescence quickly and maintains its brightness, therefore providing a reliable signal for the 48-hour cell-free reaction.

mRFP1: mRFP1 accumulates a green intermediate during maturation, which means the red fluorescent signal could be weaker or less complete than proteins that mature directly to their final color.

mKO2: mKO2 has moderate acid sensitivity, so as pH drifts over 36 hours in a cell-free reaction, its fluorescence may dim or become less reliable.

CELL-FREE REAGENTS HYPOTHESIS

Protein: mTurquoise2

Biophysical property: Slower chromophore maturation compared to sfGFP.

Reagent to adjust: Nicotinamide (tested at +48%, +100%, and +200% above baseline)

Hypothesis: mTurquoise2’s slower maturation requires sustained energy support. Increasing nicotinamide concentration will boost NAD+-dependent energy regeneration, accelerating mTurquoise2’s chromophore maturation during the 36-hour incubation and increasing fluorescence output.

Expected outcome: mTurquoise2 wells with increased nicotinamide will show brighter fluorescence than the baseline control, with fluorescence peaking at an optimal concentration before potentially declining at the highest dose. This would demonstrate that slower-maturing proteins benefit from enhanced energy support, suggesting the Ginkgo/OpenAI master mix – optimized for sfGFP – is not universally optimal for all fluorescent proteins.

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