week 11 Building genomes

HTGAA 1536 Pixel Artwork Canvas – Collective Bioart Experiment

As part of Week 11, I participated in the HTGAA 1536 Pixel Artwork Canvas, a collective bioart experiment where each participant could contribute at least one pixel to a shared global artwork. The artwork was connected to cell-free reaction compositions, where each pixel represented a small contribution to a larger collaborative biological and visual system.

For my contribution, I added pixels to the shared canvas as part of the collective image composition. I enjoyed the idea that many small individual actions could come together to create a larger community artwork. This made the project feel playful, experimental, and collaborative, while also connecting visual design with biological systems and cell-free expression.

What I liked most about the project was the combination of art, biology, and community participation. It was interesting to see how a simple pixel-based interface could represent a much larger experiment involving biological reagents, reaction design, and collective authorship.

Cell-Free Master Mix Composition: Component Roles

E. coli Lysate

BL21 (DE3) Star Lysate, including T7 RNA Polymerase
The lysate provides the biological machinery needed for transcription and translation, including ribosomes, tRNAs, enzymes, and metabolic components from E. coli. Because it includes T7 RNA polymerase, it can efficiently transcribe DNA templates controlled by a T7 promoter into mRNA for protein expression.

Salts / Buffer

Potassium Glutamate
Potassium glutamate helps recreate an intracellular-like ionic environment for the cell-free reaction. It supports proper ribosome function, protein folding, and overall enzyme activity during transcription and translation.

HEPES-KOH pH 7.5
HEPES-KOH acts as a buffer to maintain the reaction at a stable pH around 7.5. This is important because transcription, translation, and enzyme activity are sensitive to pH changes during incubation.

Magnesium Glutamate
Magnesium ions are essential cofactors for ribosome function, nucleotide interactions, and many enzymatic reactions in cell-free protein synthesis. The glutamate counterion also helps maintain a biologically compatible salt environment.

Potassium Phosphate Monobasic
Potassium phosphate monobasic contributes phosphate ions and helps support the buffering capacity of the reaction. It also participates in maintaining the correct phosphate balance needed for energy metabolism and nucleotide-related reactions.

Potassium Phosphate Dibasic
Potassium phosphate dibasic works together with the monobasic form to create a phosphate buffer system. The balance between monobasic and dibasic phosphate helps stabilize pH and supports long-duration cell-free reactions.

Energy / Nucleotide System

Ribose
Ribose provides a sugar precursor that can be used by enzymes in the lysate to regenerate nucleotide monophosphates and support energy metabolism. In the 20-hour system, it helps sustain long-term protein production more gradually than direct high-energy substrates.

Glucose
Glucose serves as a metabolic energy source that can be processed by enzymes in the lysate to help regenerate ATP and other energy carriers. This supports longer incubation times by feeding the reaction’s internal energy regeneration pathways.

AMP
AMP is a nucleotide monophosphate that can be converted into higher-energy nucleotide forms needed for RNA synthesis and energy cycling. It contributes to the nucleotide pool required for transcription and sustained reaction activity.

CMP
CMP provides the cytidine nucleotide precursor needed for RNA synthesis. During the reaction, it can be converted into CTP, which is incorporated into mRNA during transcription.

GMP
GMP provides the guanosine nucleotide precursor needed for RNA synthesis. It can be converted into GTP, which is used in transcription and also plays roles in translation-related energy processes.

UMP
UMP provides the uridine nucleotide precursor needed for RNA synthesis. It can be converted into UTP, which is incorporated into mRNA during transcription.

Guanine
Guanine acts as an additional nucleobase precursor that can support nucleotide regeneration pathways. It helps maintain the supply of guanine-containing nucleotides during longer cell-free reactions.

Translation Mix: Amino Acids

17 Amino Acid Mix
The 17 amino acid mix supplies most of the amino acids required to build the expressed protein. These amino acids are used by ribosomes during translation to assemble the polypeptide chain.

Tyrosine
Tyrosine is added separately because it can have solubility or stability limitations in amino acid mixtures. Providing it separately helps ensure enough tyrosine is available for protein synthesis.

Cysteine
Cysteine is also added separately because it is chemically reactive and can be unstable in solution. It is important for proteins that require cysteine residues, including those that may form disulfide bonds or need specific structural features.

Additives

Nicotinamide
Nicotinamide supports cofactor-related metabolism and may help maintain the activity of enzymes involved in energy regeneration. In long-duration cell-free systems, it can contribute to sustaining reaction performance over time.

Backfill

Nuclease-Free Water
Nuclease-free water is used to bring the reaction to the correct final volume without introducing enzymes that could degrade DNA or RNA. It ensures that the concentrations of all components are adjusted accurately while protecting the genetic template and transcripts.

Main Differences Between the 1-Hour PEP-NTP Mix and the 20-Hour NMP-Ribose-Glucose Mix

The 1-hour optimized PEP-NTP master mix uses high-energy components such as PEP and NTPs directly, making it suitable for fast protein expression over a short incubation time. In contrast, the 20-hour NMP-Ribose-Glucose system uses nucleotide monophosphates, ribose, and glucose to regenerate energy and nucleotides more gradually through enzymatic pathways in the lysate.

This makes the 20-hour system more sustainable and cost-effective for longer fluorescent protein production, while the 1-hour system is more immediate but likely less suitable for extended incubation.

Fluorescent Protein Properties Relevant to Cell-Free Expression

sfGFP
sfGFP, or superfolder GFP, is useful in cell-free systems because it folds efficiently and matures rapidly compared with many other GFP variants. This strong folding behavior can improve fluorescence readout even when protein expression conditions are not ideal. oai_citation:0‡FPbase

mRFP1
mRFP1 is a red fluorescent protein that is monomeric and relatively acid tolerant, but it is somewhat slow to mature and has lower brightness compared with newer red fluorescent proteins. In a cell-free system, this means fluorescence may appear later or be weaker even if protein translation is successful. oai_citation:1‡FPbase

mKO2
mKO2 is an orange fluorescent protein with good photostability and red-shifted emission, but it has moderate acid sensitivity and a maturation time of around 108 minutes. In long cell-free reactions, maintaining pH is important so that the fluorescent signal is not reduced by acidification of the reaction mixture. oai_citation:2‡PMC

mTurquoise2
mTurquoise2 is a cyan fluorescent protein reported to mature rapidly and have very low acid sensitivity. These properties make it suitable for cell-free expression because fluorescence can develop relatively quickly and remain more stable if the reaction pH changes slightly. oai_citation:3‡FPbase

mScarlet-I
mScarlet-I is a bright red fluorescent protein that is reported to be rapidly maturing, but it still has moderate acid sensitivity. In a 36-hour incubation, buffering capacity may strongly affect the final red fluorescence intensity. oai_citation:4‡FPbase

Electra2
Electra2 is a blue fluorescent protein derived from Entacmaea quadricolor. As with other fluorescent proteins, its final fluorescence depends on correct folding and chromophore maturation; blue fluorescent proteins can be more challenging to read out clearly because they may have lower brightness or require careful excitation and detection settings. oai_citation:5‡FPbase

Hypothesis for Improving Fluorescence Over a 36-Hour Incubation

Hypothesis:
For mScarlet-I, increasing the buffering capacity of the 36-hour artwork master mix by optimizing HEPES-KOH pH 7.5 and the potassium phosphate monobasic/dibasic buffer system will help maintain a stable pH during long incubation. Because mScarlet-I has moderate acid sensitivity, better pH stability should reduce fluorescence loss and improve the final red signal after 36 hours.

A possible adjustment would be to keep the reaction close to pH 7.5 by testing slightly higher HEPES-KOH and phosphate buffer concentrations while maintaining magnesium and potassium levels compatible with translation. The expected effect is stronger and more stable fluorescence because the protein can fold, mature, and remain fluorescent under less acidic reaction conditions.

Short Note for the Next Experimental Phase

In the next phase, the exact reagent concentrations for the assigned artwork wells should be chosen based on the fluorescent protein in each well. For proteins with acid sensitivity, such as mKO2 and mScarlet-I, buffer optimization should be prioritized; for slower-maturing proteins such as mRFP1, the reaction should support long-term energy regeneration and oxygen availability to allow chromophore maturation over the full 36-hour incubation.

The final phase of this lab will be analyzing the fluorescence data we collect to determine whether we can draw any conclusions about favorable reagent compositions for our fluorescent proteins. This will be due a week after the data is returned (date TBD!). The reaction composition for each well will be as follows:

6 μL of Lysate 10 μL of 2X Optimized Master Mix from above 2 μL of assigned fluorescent protein DNA template 2 μL of your custom reagent supplements Total: 20 μL reaction