Week 12 HW: Bioproduction

The homework requirements are posted under Week 11 homework page.

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

Mine didn’t work well, and I missed the April 19 due date.

Part B: Cell-Free Protein Synthesis | Cell-Free Reagents (used GenAI for answers)

  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 component’s role is in the cell-free reaction.

E. coli Lysate:

BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): Provides the core cellular machinery for transcription and translation, including ribosomes, enzymes, and T7 RNA polymerase for high-efficiency expression of genes under T7 promoters.

Salts/Buffer:

Potassium Glutamate: Maintains ionic strength and mimics intracellular conditions to support enzyme activity and protein synthesis.

HEPES-KOH pH 7.5: Acts as a buffering agent to stabilize pH during the reaction, ensuring optimal enzyme function.

Magnesium Glutamate: Essential cofactor for ribosomes and polymerases, supporting transcription and translation processes.

Potassium phosphate monobasic / Potassium phosphate dibasic: Provide additional buffering capacity and help maintain phosphate balance for energy metabolism and nucleic acid stability.

Energy / Nucleotide System:

Ribose / Glucose: Serve as energy sources and metabolic substrates to regenerate ATP and sustain the reaction.

AMP / CMP / GMP / UMP: Nucleotide monophosphates that are phosphorylated to form NTPs required for RNA synthesis.

Guanine: A nucleotide base that supports nucleotide pool balance and efficient transcription.

Translation Mix (Amino Acids):

17 Amino Acid Mix: Provides most of the amino acids required for protein synthesis.

Tyrosine / Cysteine: Added separately due to stability or solubility limitations, completing the full set of amino acids for translation.

Additives:

Nicotinamide: Acts as a cofactor precursor (NAD⁺ related), supporting redox balance and metabolic reactions in the lysate.

Backfill:

Nuclease Free Water: Used to adjust reaction volume and maintain purity by preventing degradation of 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 PEP-NTP relies on rapic but short-lived protein synthesis. The 20-hour-NMP-ribose-glucose mix relies on metabolic pathways, so it is more sustainable, though slower.

Part C: Planning the Global Experiment | Cell-Free Master Mix Design (used GenAI for answers)

  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: Engineered for enhanced folding efficiency and stability, sfGFP folds reliably even in suboptimal conditions, making it highly robust in cell-free systems.

mRFP1: Has a slow maturation time, meaning fluorescence develops gradually, which can limit early signal detection in short reactions.

mKO2: Sensitive to pH changes (acid sensitivity), so fluorescence intensity can decrease if the reaction environment becomes slightly acidic over time.

mTurquoise2: Requires proper protein folding for high quantum yield, and misfolding can significantly reduce fluorescence output.

mScarlet_I: Optimized for fast maturation and high brightness, making it well-suited for strong fluorescence but still dependent on proper folding conditions.

Electra2: A newer fluorescent protein with enhanced brightness but potential sensitivity to oxygen and folding conditions, which can impact fluorescence consistency.

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

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: Increasing magnesium glutamate concentration and optimizing energy regeneration (e.g., glucose-based system) will improve folding efficiency and sustained expression of mRFP1, leading to higher fluorescence over 36 hours.

Protein: mRFP1

Reagents: Increased magnesium glutamate concentration and a glucose-based energy regeneration system

    1. I don’t think these applies to me, as I don’t have the lab equipment nor received an email instruction. Expected Effect: Enhancing magnesium levels will improve ribosome activity and protein folding, while sustained energy supply from glucose will extend protein synthesis time, allowing more mRFP1 to properly fold and mature. This will result in higher fluorescence intensity over a 36-hour incubation.