Week 11 — Bioproduction & Cloud Labs

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

  • what you contributed to the community bioart project I tried to transform a hexagon into a bacteriophage by adding some details in the exterior area. I think they were restored to the original picture before deadline
  • what you liked about the project I was reminded of another collaborative project that was funny
  • what about this collaborative art experiment could be made better for next year. Maybe a bigger canvas

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

  1. 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 components role is in the cell-free reaction.

BL21 (DE3) Star Lysate (includes T7 RNA Polymerase):

Provides the complete transcription translation machinery (ribosomes, tRNAs, enzymes) required for protein synthesis; the incorporated T7 RNA polymerase enables high- fficiency transcription from T7 promoters.

Potassium Glutamate

Maintains intracellular-like ionic strength and stabilizes ribosome structure, improving translation efficiency.

HEPES-KOH pH 7.5

Acts as a buffering agent to maintain a stable physiological pH optimal for enzymatic activity during transcription and translation.

Magnesium Glutamate

Supplies Mg²⁺ ions, which are essential cofactors for ribosome assembly, ATP utilization, and nucleic acid stability.

Potassium phosphate monobasic

Contributes to buffering capacity and provides phosphate ions required for nucleotide metabolism.

Potassium phosphate dibasic

Works with the monobasic form to stabilize pH and maintain phosphate balance for energy transfer reactions.

Ribose: Serves as a precursor for nucleotide biosynthesis and contributes to maintaining energy metabolism.

Glucose: Functions as a primary energy source, fueling ATP regeneration through glycolytic enzymes present in the lysate.

AMP: Acts as a nucleotide precursor and participates in energy recycling pathways within the system.

CMP: Provides cytidine nucleotides required for RNA synthesis.

GMP: Supplies guanosine nucleotides necessary for transcription and translation processes.

UMP: Contributes uridine nucleotides for RNA synthesis.

Guanine: Serves as an additional base precursor to support nucleotide pool balance and synthesis.

17 Amino Acid Mix: Provides the majority of amino acids required for protein synthesis, excluding those prone to instability or oxidation.

Tyrosine: Supplied separately due to limited solubility, ensuring sufficient availability for incorporation into proteins.

Cysteine: Added independently because of its susceptibility to oxidation, maintaining proper redox conditions for protein synthesis.

Nicotinamide: Functions as a precursor for NAD⁺/NADH, supporting redox balance and metabolic reactions necessary for sustained protein synthesis.

Nuclease Free Water: Adjusts the final reaction volume and maintains reagent concentrations without introducing nucleases that could degrade DNA or RNA.

  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 1 hour PEPNTP system uses direct high-energy substrates (PEP) and fully supplied NTPs, enabling rapid transcription and translation but with fast energy depletion and shorter reaction lifetimes. In contrast, the 20 hour NMP ribose glucose system relies on metabolic regeneration, where nucleotides are built from NMPs and ribose and ATP is regenerated via glucose driven pathways, supporting longer, more sustainable protein synthesis. Additionally, the 20-hour system is simplified and more balanced (fewer additives, inclusion of phosphate buffering and nicotinamide), prioritizing stability and longevity over the high initial reaction speed seen in the PEP-based mix.

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.

sfGFP

Superfolder GFP is engineered for enhanced folding robustness, allowing efficient chromophore formation even under suboptimal conditions typical of cell-free systems. This makes it one of the most reliable reporters with strong signal output.

mRFP1

mRFP1 has a relatively slow maturation time, meaning fluorescence appears later after translation. In CFPS, this can lead to underestimation of expression at early timepoints.

mKO2

mKO2 is acid-sensitive, with fluorescence decreasing at lower pH. Since CFPS reactions can acidify over time due to metabolism, its signal may diminish during long incubations.

mTurquoise2

mTurquoise2 has a high quantum yield and efficient chromophore formation, producing bright fluorescence even at lower protein concentrations. This improves sensitivity in CFPS readouts.

mScarlet_I

mScarlet I is optimized for fast maturation among red fluorescent proteins, enabling earlier fluorescence detection compared to older RFPs. This is advantageous for time-course measurements in CFPS.

Electra2

Electra2 is oxygen dependent for chromophore formation, like most fluorescent proteins. Limited oxygen availability in CFPS (especially in closed reactions) can reduce or delay fluorescence development.

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 mKO2, increasing the HEPES-KOH buffer concentration and optimizing the phosphate buffer ratio in the 36 hour mastermix will better maintain pH near 7.5, reducing acid driven loss of fluorescence during long incubation. Expected effect: Because mKO2 fluorescence is acid sensitive, stronger buffering should preserve chromophore brightness and produce a higher final fluorescent signal over 36 hours.