Week 11 HW

Homework — Due by Start of Apr 28 Lecture

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

I contributed one pixel to the global artwork experiment. My contribution was intentionally small, but I liked that the project made each person’s local decision become part of a larger shared image. The final artwork depended on many tiny choices accumulating together rather than on one centralized author.

What I liked most was the relationship between scale and authorship. A single pixel is almost invisible on its own, but within the full canvas it becomes part of a collective biological image. That felt appropriate for a bioart project because the artwork behaved almost like a living system: many small inputs produced an emergent pattern that no individual participant could fully control.

For next year, the project could be improved by making the collaboration process more visible. A time-lapse showing how the artwork changed over time would help viewers understand the collective process behind the final image. It could also be interesting to let contributors leave short notes about their choices, so the project records both the final visual outcome and the distributed decisions that produced it.

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

Roles of each component

E. coli Lysate

BL21(DE3) Star Lysate, including T7 RNA Polymerase
The lysate provides the core biological machinery for transcription and translation, including ribosomes, tRNAs, aminoacyl-tRNA synthetases, translation factors, metabolic enzymes, and other cellular components. Because it includes T7 RNA polymerase, it can efficiently transcribe DNA templates under a T7 promoter.

Salts / Buffer

Potassium Glutamate
Potassium glutamate helps recreate the ionic environment of the bacterial cytoplasm. Potassium supports protein synthesis, while glutamate is a biologically compatible counterion that helps maintain reaction performance.

HEPES-KOH, pH 7.5
HEPES-KOH buffers the reaction near physiological pH. This is important because transcription, translation, enzyme activity, and fluorescent protein stability are all sensitive to pH changes.

Magnesium Glutamate
Magnesium is essential for ribosome structure, tRNA function, nucleotide chemistry, and many enzymes in transcription and translation. Magnesium concentration must be tuned carefully because too little reduces translation, while too much can inhibit or destabilize the system.

Potassium Phosphate Monobasic
Potassium phosphate monobasic contributes phosphate and buffering capacity. Together with the dibasic form, it helps maintain pH and supports phosphate-dependent energy metabolism.

Potassium Phosphate Dibasic
Potassium phosphate dibasic pairs with the monobasic phosphate to create a phosphate buffer system. It also contributes potassium ions and phosphate needed for metabolic and energy-regeneration reactions.

Energy / Nucleotide System

Ribose
Ribose provides a sugar backbone that can support nucleotide regeneration and metabolic activity. In long-duration reactions, it helps the system rebuild nucleotide triphosphates rather than relying only on pre-supplied NTPs.

Glucose
Glucose is an energy substrate that can be metabolized by enzymes in the lysate. It supports ATP regeneration over longer reactions.

AMP
AMP is a nucleotide precursor that can be converted into ATP through phosphorylation pathways. It supports a lower-cost, regenerating energy system.

CMP
CMP is a precursor for CTP regeneration. It supports RNA synthesis once converted into the triphosphate form.

GMP
GMP is a precursor for GTP regeneration. GTP is needed for transcription and also plays a major role in translation.

UMP
UMP is a precursor for UTP regeneration. UTP is required for RNA synthesis during transcription.

Guanine
Guanine is a nucleobase that can enter nucleotide salvage pathways. It can be converted into guanine nucleotides and eventually support GTP production.

Translation Mix / Amino Acids

17 Amino Acid Mix
The amino acid mix provides most of the building blocks needed to synthesize the target protein. Without sufficient amino acids, translation stops or produces low yield.

Tyrosine
Tyrosine is supplied separately because it has solubility and stability constraints compared with many other amino acids. It is also especially relevant for fluorescent proteins, since aromatic residues help form the chromophore environment.

Cysteine
Cysteine is often handled separately because it is chemically reactive and can oxidize. Maintaining cysteine availability helps prevent amino acid limitation during protein synthesis.

Additives

Nicotinamide
Nicotinamide supports metabolic cofactor balance, especially pathways related to NAD/NADH chemistry. In a cell-free system, maintaining cofactor availability helps sustain energy metabolism and enzyme activity.

Backfill

Nuclease-Free Water
Nuclease-free water is used to bring the reaction to the final volume without introducing nucleases that could degrade DNA or RNA. It is the neutral backfill component for controlling final reaction concentration.

Difference between the 1-hour optimized PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix

The 1-hour optimized PEP-NTP master mix is designed for fast, high-intensity expression by directly supplying NTPs and using phosphoenolpyruvate (PEP) as a strong energy-regeneration substrate. It is useful when the goal is rapid protein production over a short time window.

The 20-hour NMP-Ribose-Glucose master mix is designed for longer reactions by using nucleotide monophosphates, ribose, and glucose to regenerate nucleotides and ATP over time. It is slower but more sustained, making it better for long incubations such as overnight or 36-hour fluorescence development.

Bonus: How can transcription occur if GMP is not included but Guanine is?

Transcription can still occur because guanine can enter nucleotide salvage pathways. The lysate can convert guanine into GMP, then phosphorylate GMP into GDP and GTP. GTP can then be used by RNA polymerase during transcription.

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

Fluorescent protein properties affecting cell-free expression or readout

sfGFP

sfGFP is useful in cell-free systems because it folds and matures quickly and is engineered to tolerate imperfect folding environments. Its main limitation is that fluorescence still requires chromophore maturation, which depends on oxygen and time.

mRFP1

mRFP1 matures faster than the older DsRed protein, but it has lower brightness and photostability than some newer red fluorescent proteins. In a cell-free reaction, this means it may need longer incubation or stronger expression to produce a visible signal.

mKO2

mKO2 is an orange fluorescent protein with moderate acid sensitivity. This means the readout may be affected if the cell-free reaction becomes acidic during long incubation.

mTurquoise2

mTurquoise2 is a cyan fluorescent protein known for strong performance in imaging, including relatively good photostability. In cell-free systems, the main concern is making sure the excitation/emission settings match cyan fluorescence and that the protein has enough time and oxygen to mature.

mScarlet_I

mScarlet_I is a bright monomeric red fluorescent protein with accelerated maturation compared with mScarlet. However, red fluorescent proteins can still be sensitive to maturation time, oxygen availability, and pH during long cell-free incubation.

Electra2

Electra2 is a blue fluorescent protein with low pKa, which suggests relatively strong resistance to acid quenching. Its readout may still be harder to detect than green or red proteins because blue fluorescence can be more sensitive to instrument settings, background, and excitation conditions.

Hypothesis for improving fluorescence over a 36-hour incubation

Protein: mScarlet_I

Reagents to adjust: HEPES-KOH, potassium phosphate buffer, magnesium glutamate, amino acid mix, tyrosine, cysteine, and glucose/ribose energy system.

Hypothesis:
For mScarlet_I, increasing buffer capacity with HEPES-KOH and phosphate while maintaining an optimized magnesium glutamate concentration will improve fluorescence over 36 hours by stabilizing pH and supporting ribosome activity. Adding extra amino acid mix, especially tyrosine and cysteine, could prevent amino acid depletion during long translation. Using the NMP-Ribose-Glucose energy system should support sustained ATP and NTP regeneration, allowing protein production and chromophore maturation to continue over a longer time window.

Expected effect:
The reaction should produce a stronger final red fluorescence signal after 36 hours because the system remains active for longer, avoids pH-related fluorescence loss, and maintains enough amino acid and energy supply for continued protein synthesis.

Part D: Build-A-Cloud-Lab | Optional Bonus Assignment

For the optional cloud lab design, I would create a circular, modular “biofoundry island” made of Ginkgo Reconfigurable Automation Carts. The center would contain shared analysis equipment, while the outer ring would contain specialized carts for liquid handling, incubation, imaging, and sample storage.

This layout would make the lab feel less like a linear factory and more like a flexible organism. Samples could move around the ring depending on the workflow, and individual carts could be swapped or reconfigured without changing the whole system. The design would be especially useful for experiments like cell-free synthesis, where many small reactions need to be assembled, incubated, imaged, and compared in parallel.

References