Week 11 HW: Bioproduction & Cloud Labs
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.
Done.
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.
I contributed a little dot on the bottom right plate, but it was overlapped by other contributions later. I liked the interactivity of the project. It’s cool to see how the plates evolve over time as more people contribute.
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 component’s role is in the cell-free reaction.
- E. coli Lysate: Provides the necessary cellular machinery (e.g., ribosomes, tRNAs, enzymes) for transcription and translation to occur in the cell-free system.
- Salts/Buffer: Provide suitable pH conditions for cell-free reactions.
- Energy / Nucleotide System: Provide energy sources needed for the reactions.
- Translation Mix (Amino Acids): Provide the building blocks for protein synthesis.
- Additives (Nicotinamide): Improve the efficiency of the reactions.
- Backfill (Nuclease Free Water): Adjust the final volume of the reaction and ensure that it is nuclease-free to prevent degradation of DNA/RNA.
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)
1-hour optimized PEP-NTP: Rapid protein synthesis, using phosphoenolpyruvate (PEP) as an energy source.
20-hour NMP-Ribose-Glucose master mix: Optimized for longer reactions, using nucleoside monophosphates (NMPs) and glucose for sustained energy production over a longer period.
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
- Maturation Time: 5–15 minutes, very fast
- Acid Sensitivity: generally more stable, maintains roughly 50% of its fluorescence at pH 5.4
- Folding: high folding efficiency, fluorescence is thus enhanced in cell-free systems
- Oxygen Dependence: oxygen required
2. 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.
Use glucose as the main energy source in the master mix to sustain energy production over a longer period, which could improve the folding and maturation of sfGFP, leading to increased fluorescence over a 36-hour incubation.