Week 11: BUILDING GENOMES

openimage openimage

Week # 11 Building Genomes

BUILDING GENOMES

To inspire collaboration and creativity while designing a scientifically rigorous cell-free fluorescent protein optimization experiment together.

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

1. Contribute at least one pixel to this global artwork experiment before the editing ends on Sunday 4/19 at 11:59 PM EST.
    ◦ A personalized URL was sent to the email address associated with your Discourse account, and you can discuss the artwork on the Discourse.

https://rcdonovan.com/synbiobeta I contributed 3 on the in the middle of the artwork

    ◦ If you did not have a chance to contribute, it’s okay, just make sure you become a TA this fall! 😉
2. Make a note on your HTGAA webpages including:
    ◦ what you contributed to the community bioart project (e.g., “I made part of the DNA on the bottom right plate”)
    ◦ what you liked about the project, and
    ◦ what about this collaborative art experiment could be made better for next year.

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

  Referencing the cell-free protein synthesis reaction composition (the middle box outlined in yellow on the image above, also listed below), provide a 1-2 sentence description of what each component’s role is in the cell-free reaction.

E. coli Lysate

  • BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): - Provides the cellular machinery needed for transcription and translation, including ribosomes, elongation factors, enzymes, and T7 RNA polymerase to drive expression from T7 promoters.

Salts and Buffer

  • Potassium Glutamate: - Maintains ionic strength and osmotic balance, helping transcription and translation machinery function properly. -HEPES-KOH pH 7.5: - Buffers the reaction to keep pH stable, which is important for enzyme activity and protein synthesis.
  • Magnesium Glutamate: Supplies magnesium, a key cofactor that stabilizes ribosomes, nucleic acids, and many reaction enzymes.
  • Potassium phosphate monobasic: Contributes phosphate buffering and can help support energy-related chemistry in the reaction.
  • Potassium phosphate dibasic: Works with the monobasic form to set and maintain the desired pH and buffering capacity.

Energy and Nucleotides

  • Ribose: Provides a sugar backbone component for nucleotide-related chemistry and can support metabolic regeneration pathways.
  • Glucose: Serves as an energy source to help regenerate ATP and sustain the reaction.
  • AMP: Participates in energy metabolism and regeneration pathways that help maintain usable nucleotide pools.
  • CMP: Supports the nucleotide pool needed for RNA synthesis.
  • GMP: Supports the nucleotide pool needed for RNA synthesis.
  • UMP: Supports the nucleotide pool needed for RNA synthesis.
  • Guanine: Contributes to nucleotide salvage and replenishment of GTP-related pools.

Translation Mix

  • 17 Amino Acid Mix: Supplies the protein-building substrates needed for translation into the target protein.
  • Tyrosine: Provides an additional amino acid component for protein synthesis.
  • Cysteine: Provides an additional amino acid component for protein synthesis and can be important for protein structure via disulfide bonds.

Additives

  • Nicotinamide: Supports NAD-related metabolic recycling, helping sustain energy generation in the reaction.

Backfill

  • Nuclease Free Water: Brings the reaction to final volume while avoiding nucleases that could degrade DNA or RNA templates.
  1. 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 main difference is that the PEP-NTP master mix is designed for a fast, short reaction and uses phosphoenolpyruvate as the primary energy source, while the NMP-Ribose-Glucose master mix is built for a longer reaction and uses simpler metabolic substrates to sustain activity over many hours. In practical terms, the 1-hour mix favors rapid output, whereas the 20-hour mix favors longer-lasting protein synthesis and template usage.

Energy strategy

PEP-based systems usually generate energy more directly and quickly, which helps drive early, high-rate transcription and translation. NMP-Ribose-Glucose systems rely more on gradual metabolic recycling and downstream regeneration, so they tend to support a longer reaction window.

Reaction duration

The 1-hour optimized mix is tuned for speed, so it is useful when you want a quick readout. The 20-hour mix is tuned for persistence, so it is better when you want prolonged expression or higher total yield over time.

Practical implication

If you need a fast assay or screening result, the PEP-NTP mix is usually the better fit. If you need the reaction to keep running much longer, the NMP-Ribose-Glucose mix is the more suitable choice.

  1. Bonus question: How can transcription occur if GMP is not included but Guanine is? Transcription can still occur because guanine is a precursor, not the RNA building block itself. In the lysate, enzymes can convert guanine into GMP, then to GDP and GTP, and GTP is the actual nucleotide that RNA polymerase uses to build RNA.

Why guanine is enough

Free guanine can be salvaged into the nucleotide pool through the cell-free extract’s metabolic enzymes, so the reaction does not need GMP to be added separately. Once GMP is made, it can be phosphorylated to the triphosphate form needed for transcription.

What transcription needs

RNA synthesis requires the four ribonucleoside triphosphates: ATP, CTP, GTP, and UTP. So the important point is not whether GMP is added directly, but whether the system can maintain enough GTP availability for RNA polymerase to work.

In your mix

The presence of guanine suggests the master mix is designed to replenish guanine nucleotides metabolically rather than supplying GMP outright. That is consistent with a longer-running cell-free system that relies on internal recycling and salvage pathways.

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

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

mRFP1

mKO2

mTurquoise2

mScarlet_I

Electra2

The amino acid sequences are shown in the HTGAA Cell-Free Benchling folder.

Cell-free protein synthesis (CFPS) offers a unique environment for expressing fluorescent proteins (FPs). Because these systems lack the complex regulatory machinery of a living cell, the intrinsic biophysical properties of the protein itself—such as how fast it folds or how much oxygen it requires—become the primary drivers of the visible signal.

  1. sfGFP (Superfolder GFP)

Property: Folding Robustness

sfGFP was specifically engineered to resist misfolding, allowing it to reach its functional state even when fused to poorly folding proteins or expressed at high speeds in cell-free systems. This makes it the “gold standard” for CFPS because it ensures that nearly all translated protein becomes fluorescently active rather than forming insoluble aggregates.

  1. mRFP1 (Monomeric Red Fluorescent Protein 1)

Property: Maturation Time

As a first-generation monomeric red FP, mRFP1 suffers from a relatively slow maturation time (often exceeding one hour). In cell-free reactions, this creates a significant “time lag” between protein synthesis and signal detection, which can obscure the real-time dynamics of genetic circuits.

  1. mKO2 (Monomeric Kusabira Orange 2)

Property: Acid Sensitivity (pK_a)

mKO2 is sensitive to pH changes, with a pK_a of approximately 5.0. While cell-free buffers are usually stable, the accumulation of metabolic byproducts (like organic acids) during long reactions can drop the pH, potentially quenching the mKO2 signal and leading to an underestimation of protein yield.

  1. mTurquoise2

Property: Quantum Yield (Brightness)

mTurquoise2 possesses an exceptionally high quantum yield (0.93), making it one of the brightest cyan FPs available. This high intrinsic brightness allows for a very high signal-to-noise ratio in cell-free readouts, which is critical when working with low-yield reactions or microfluidic droplets where the total amount of protein is minimal.

  1. mScarlet-I

Property: Maturation Kinetics

mScarlet-I is designed for high brightness and fast maturation (t_{1/2} \approx 36 minutes), which is significantly faster than many other red FPs. This rapid maturation makes it an ideal reporter for cell-free systems where users need to observe red fluorescence almost immediately after the start of translation.

  1. Electra2

Property: Oxygen Dependence / Fast Maturation

Electra2 was specifically developed for ultra-fast readouts in time-resolved applications. Its primary functional advantage in cell-free systems is its optimized maturation speed, which minimizes the delay in signal acquisition for high-throughput screening and the characterization of rapid transcriptional-translational (TX-TL) bursts.

sfGFP folds rapidly and efficiently even under suboptimal conditions due to its superfolder mutations, making it ideal for cell-free systems where chaperone activity is limited. Its low pKa (around 3.1) ensures stable fluorescence across a wide pH range typical in these reactions.

mRFP1 has a relatively slow maturation time (around 60 minutes), which delays fluorescence readout in time-sensitive cell-free assays. It also shows moderate folding efficiency, potentially leading to lower yields in crowded lysate environments.

mKO2 exhibits fast maturation and high quantum yield (0.62), allowing quick orange fluorescence detection in cell-free setups. However, its slightly higher pKa (5.5) makes it more pH-sensitive than GFP variants, risking signal loss if the reaction acidifies.

mTurquoise2 matures very rapidly (half-time ~33 minutes) with minimal acid sensitivity (pKa 3.1), providing bright cyan signal early and reliably in cell-free transcription-translation. Its monomeric state prevents aggregation issues that plague some FPs in cell-free crowding.

mScarlet_I offers extremely fast maturation (<15 minutes reported in literature) and high photostability, enabling high-throughput cell-free screening with red-shifted emission. Its optimized folding minimizes misfolding in oxygen-variable cell-free conditions.

Electra2 (a newer far-red FP) shows oxygen-independent chromophore formation like other iLOV-derived proteins, crucial for anaerobic cell-free reactions. Its rapid folding supports expression monitoring without oxygenation dependence that hampers traditional FPs.

References

  1. The use of LLM models in research and reporting