Week 11 HW: Building genomes

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

Make a note on your HTGAA webpages including:

  1. what you contributed to the community bioart project (e.g., “I made part of the DNA on the bottom right plate”) I made 3 dots on the lower right quadrant. In green.

  2. what you liked about the project It was something new and also interesting to see how creative people can be

  3. what about this collaborative art experiment could be made better for next year. People discovered how to beat the system and be able to contribute more than once. So by the time I was joining alot of the spots had already been filled. Next year it would be nice for maybe the team to know what art work they would like done so that one can know how best to contribute. But all in all it was a new unique experience.

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

1. Role of each component:

E. coli Lysate (BL21 DE3 Star + T7 RNA Polymerase) Provides all the cellular machinery needed for transcription and translation — ribosomes, tRNA, elongation factors, and T7 RNAP to transcribe DNA into mRNA. Salts/Buffer:

Potassium Glutamate — Mimics intracellular ionic conditions; supports ribosome function and enzyme activity. HEPES-KOH pH 7.5 — Maintains a stable physiological pH throughout the reaction. Magnesium Glutamate — Mg²⁺ is essential for ribosome assembly and stabilizing nucleic acids. Potassium Phosphate Monobasic / Dibasic — Act as a pH buffer and provide phosphate for energy metabolism.

Energy / Nucleotide System:

Ribose & Glucose — Carbon/energy sources that feed metabolic pathways regenerating ATP and other nucleotides. AMP, CMP, GMP, UMP — Nucleoside monophosphates used as building blocks for RNA synthesis (transcription). Guanine — A nucleobase that can be salvaged and converted to GTP, supporting transcription without requiring pre-formed GMP to be included directly.

Translation Mix (Amino Acids):

17 Amino Acid Mix, Tyrosine, Cysteine — The complete set of 20 amino acids needed for protein synthesis. Tyrosine and Cysteine are kept separate due to their poor solubility or tendency to oxidize.

Nicotinamide — A precursor to NAD⁺; supports cellular redox reactions and energy regeneration.

Nuclease-Free Water — Brings the reaction to the correct final volume without introducing RNases that would degrade mRNA.

2. 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 1-hour PEP-NTP mix uses phosphoenolpyruvate (PEP) as a fast energy source and pre-formed nucleoside triphosphates (NTPs), enabling rapid but short-lived transcription/translation. The 20-hour NMP-Ribose-Glucose mix uses nucleoside monophosphates plus ribose and glucose as slower-releasing energy sources, allowing the reaction to sustain itself over a much longer incubation. The tradeoff is speed vs. duration — PEP-NTP is optimized for quick protein yield, while NMP-Ribose-Glucose is designed for extended, lower-intensity expression.

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

Guanine can be salvaged by the cell-free system’s enzymes and converted to GMP, then GDP, and finally GTP through phosphorylation steps driven by the energy system in the mix. So transcription can still proceed because GTP is synthesized in situ from the guanine base.

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

  1. 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 — Superfolder GFP is engineered for extremely robust and rapid folding, making it highly tolerant to misfolding conditions; it matures quickly and fluoresces reliably even in cell-free environments.
  • mRFP1 — mRFP1 has a relatively slow chromophore maturation time and requires oxygen for oxidation of the chromophore, which can limit its fluorescence in oxygen-limited cell-free reactions.
  • mKO2 — mKO2 (monomeric Kusabira Orange 2) has a moderate maturation rate and is sensitive to acidic pH, which can reduce fluorescence if the reaction pH drifts below 7.
  • mTurquoise2 — mTurquoise2 has a high quantum yield and fast maturation, making it well-suited for cell-free systems; it is relatively photostable and performs well over long incubations.
  • mScarlet-I — mScarlet-I is a fast-maturing red fluorescent protein with high brightness; the “I” variant was specifically engineered for improved monomericity and fast maturation compared to the original mScarlet.
  • Electra2 — Electra2 is a recently engineered protein optimized for cell-free expression; it is designed for low oxygen dependence and fast maturation, making it particularly well-suited for extended cell-free reactions.
  1. 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

Hypothesis for improving fluorescence:

  • Increase Magnesium Glutamate concentration slightly above baseline. Hypothesis: mRFP1 has slow maturation and relies heavily on correct folding of its β-barrel structure. Increasing Mg²⁺ availability can stabilize ribosome activity and improve translational fidelity, leading to more correctly folded mRFP1 and higher total fluorescence over a 36-hour incubation.

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

Skipping this. Might revist later.