Week 11 HW: Building Genomes

This page tackles all homeworks of week 11.

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

I made a long-necked tall green dinosaur with its bottom half in the fourth quadrant and its long neck and head stretching into the first quadrant; someone else (should be nicknamed Asteroid) changed those pixels and made the chaotic drawing more symmetric and distinguishing quadrants (not allowing any common information flow across the different drawing quadrants).

Instead of allowing everyone to change all pixels with a time-gated manner, I think everyone should be allowed to have their own full canvas to make the full design. Then others will be able to vote on which of those drawings could be integrated into the main canvas. Like say my dino could be 10*10 pixels and if selected it could be placed somewhere in the main canvas; or everyone’s canvas could be superimposed, or just integrate individual tiles for a mega picture!!!

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

Component Roles in the Cell-Free Reaction

• E. coli Lysate / BL21 (DE3) Star Lysate (includes T7 RNA Polymerase)
  • This is the core biological engine of the reaction. It provides the heavy machinery (like ribosomes) to read the genetic code and build proteins, while the T7 polymerase specifically transcribes the DNA template into RNA.
• Salts/Buffer (Potassium Glutamate, HEPES-KOH pH 7.5, Magnesium Glutamate, Potassium phosphate monobasic/dibasic)
  • This acts as the environmental stabilizer. It maintains a steady pH so the machinery doesn’t degrade, while the salt ions (especially magnesium) serve as essential chemical helpers that allow the ribosomes to clip together correctly.
• Energy / Nucleotide System (Ribose, Glucose, AMP, CMP, GMP, UMP, Guanine)
  • This functions as the fuel station and letter pool. The nucleotides (AMP, CMP, GMP, UMP) are the raw alphabet letters used to build RNA, while sugars like glucose and ribose are broken down to keep charging up the system’s battery.
• Translation Mix (Amino Acids) (17 Amino Acid Mix, Tyrosine, Cysteine)
  • These are the physical construction bricks. They represent the full set of 20 standard amino acids that the ribosome strings together to assemble the final physical protein chain.
• Additives (Nicotinamide)
  • This is a metabolic booster. It helps protect and stabilize key energy-carrying helper molecules in the mix, ensuring the reaction keeps running smoothly without stalling out.
• Backfill (Nuclease Free Water)
  • This is the clean liquid filler. It brings the entire mixture up to the exact required operational volume without introducing any dirty enzymes that might accidentally chew up the DNA or RNA.

PEP-NTP vs. NMP-Ribose-Glucose Master Mixes

The 1-hour optimized PEP-NTP mix uses a high-energy “rocket fuel” molecule (PEP) and pre-made premium batteries (NTPs) to create an explosive burst of protein production that burns out very quickly. In contrast, the 20-hour NMP-Ribose-Glucose mix relies on cheap, raw starter ingredients (NMPs and sugars) that force the lysate to slowly build its own energy network from scratch, resulting in a much longer, sustained marathon reaction.

Bonus Question: Transcription without GMP but with Guanine

Even though the pre-made nucleotide letter GMP is missing, the biological engine in the lysate can use a built-in salvage pathway to grab the raw base Guanine and chemically stick a ribose sugar and phosphate onto it. This transforms the plain Guanine into a fully functional nucleotide building block, allowing transcription to proceed normally.

         Phosphoribosylation (HGPRT)               Diphosphate Synthesis (GMK)                 Triphosphate Synthesis (NDK)
Guanine ──────────────────────────────▶   GMP   ────────────────────────────────▶   GDP   ─────────────────────────────────▶ GTP

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

Biophysical and Functional Properties of the 6 Fluorescent Proteins

• sfGFP (Superfolder GFP) This protein has incredibly robust, fast-folding kinetics. It folds so efficiently that it resists misfolding and aggregates much less than standard proteins, giving a very bright and reliable readout quickly.

• mRFP1 (Monomeric Red Fluorescent Protein 1) This protein suffers from relatively slow chemical maturation times and a lower quantum yield (innate brightness). Because it takes a long time for the internal chemical “lightbulb” to finish forming, its red signal can be quite faint early on.

• mKO2 (Monomeric Kusabira Orange 2) This orange protein is highly sensitive to the pH of its environment. If the reaction mixture becomes even slightly too acidic during incubation, its fluorescence output drops off sharply.

• mTurquoise2 This cyan reporter has a phenomenal quantum yield (it shines very brightly once folded), but it has complex maturation kinetics. It requires a steady supply of molecular oxygen to successfully run the internal chemical reaction that activates its fluorescence.

• mScarlet_I This is a cutting-edge red protein with superior brightness, but it is notoriously prone to photobleaching (fading under continuous light exposure). It needs to be kept in a stable, well-supported environment so its delicate structure doesn’t wear out over time.

• Electra2 This variant is explicitly engineered for rapid translation and immediate readout. However, its fast-paced assembly makes it highly dependent on a massive, uninterrupted initial pool of raw resources, so it doesn’t stall out.

Master Mix Optimization Hypothesis

Hypothesis: To maximize mRFP1 red fluorescence over a long 36-hour marathon incubation, we should supplement the custom reagent slots with an enhanced Energy Regeneration System consisting of Creatine Phosphate and Creatine Kinase (alongside a mild increase in HEPES-KOH buffer concentration).

Expected Effect: Because mRFP1 is a slow-maturing protein, a standard system will run out of batteries before the protein has time to chemically ignite its lightbulb. The custom energy supplement will continuously replenish spent ADP with ATP over the long haul, while the extra buffer prevents the system from becoming acidic, providing the sluggish mRFP1 chains with a stable, high-energy environment they need to fully mature and reach maximum brightness.

UPDATE: Also check out this HW for a better tabular answer to Parts B and C (higher readability!).

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