Week 11 HW: Bioproduction and Cloud Labs

Contents

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

I made most of the big bulls-eye target in the upper right quadrant that occurred fairly early on in the editing time period. I made it largely during the recitation just by having it open in another window from the lecture and clicking another pixel whenever my timer ran out. I didn’t really contribute after that, but it was fun to see how people incorporated the target into one of the scissors handles, and then it ultimately disappeared. For future iterations, I’d really recommend publishing the viewable history link somewhere because I lost it after like a day and so then I wasn’t able to keep watching the changes and how they compared to previous versions.

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

  • BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): Lysate includes enzymes, nutrients, and cofactors; specifically, it includes the T7 RNA polymerase for rapid transcription of genes under T7 promoter.

Salts/Buffer

  • Potassium Glutamate: Potassium glutamate is a potassium source; potassium is an essential enzyme co-factor.
  • HEPES-KOH pH 7.5: HEPES buffer maintains pH at the optimal pH for enzyme efficiency; transcription and translation usually occurs at neutral pHs which the inside of a cell is. KOH is the hydroxide to adjust buffer to pH 7.5 to because potassium phosphate is used.
  • Magnesium Glutamate: Magnesium glutamate is a magnesium source; magnesium is an essential enzyme co-factor.
  • Potassium phosphate monobasic: Potassium phosphate is both a phosphate (energy) source and a potassium (enzyme co-factor) source; I’m unsure why both the monobasic and dibasic are included here. Monobasic is mildly acidic in comparison.
  • Potassium phosphate dibasic: Potassium phosphate is both a phosphate (energy) source and a potassium (enzyme co-factor) source; I’m unsure why both the monobasic and dibasic are included here. Dibasic is mildly basic in comparison.

Energy / Nucleotide System

  • Ribose: Ribose is a sugar molecule that is an essential component of nucleic acids and ATP. It’s used in nucleotide production (GMP from guanine) and possibly also energy regernation.
  • Glucose: Glucose is a sugar molecule that is used in ATP regeneration.
  • AMP: Adenosine monophosphate is a nucleotide used in transcription. It gets additional phosphate groups to become ATP, which is essential for energy, and so it is probably also used in ATP regeneration.
  • CMP: Cytidine monophosphate is a nucleotide used in transcription.
  • GMP: Guanosine monophosphate is a nucleotide used in transcription.
  • UMP: Uridine monophosphate is a nucleotide used in transcription.
  • Guanine: Guanine is the nucleoside base for GMP; it can be used to make GMP with ribose and phosphate probably?

Translation Mix (Amino Acids)

  • 17 Amino Acid Mix: Amino acids are needed for translation because they are what proteins are made up of. I’m not sure why this is only 17 instead of 20.
  • Tyrosine: This is an amino acid needed for translation. I’m unsure why additional tyrosine would need to be added beyond the mix, maybe it’s not one of the 17?
  • Cysteine: This is an amino acid needed for translation. I’m unsure why additional cysteine would need to be added beyond the mix, maybe it’s not one of the 17?

Additives

  • Nicotinamide: Nicotinamide is part of NAD+/NADP+, and so is needed for energy regeneration in redox reactions.

Backfill

  • Nuclease Free Water: It’s an aqueous solution, so water fills out the rest of the reaction volume; nuclease-free water doesn’t contain active restriction enzymes to cleave 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.
    The main difference appears to be in energy regeneration and cheapter components, because it uses nucleotide monophosphates instead of triphosphates. It also uses PEP-Mono (phosphoenol pyruvate, monosodium salt) for energy instead of nucleotides and sugars for energy generation through enzymatic pathways like glycolysis. PEP-Mono is a high energy phosphate-containing compound that can easily transfer phosphate groups for energy.

  2. Bonus question: How can transcription occur if GMP is not included but Guanine is?
    GMP is produced with guanine, ribose, and phosphate that is provided separately in the cell-free mixture. not sure which specific enzyme(s) are involved

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.
    FPbase.org is not currently working, so i’m doing the best i can based off of skimming papers because i don’t have time to do a close reading of a whole bunch of them unfortunately. honestly, i’ll probably just try again later.
    1. sfGFP: sfGFP was developed to be faster at folding into the active, fluorescent shape than wild-type GFP, resulting in a more robust and stable fluorescent protein.
    2. mRFP1: Needs to bind to calcium?
    3. mKO2
    4. mTurquoise2
    5. mScarlet_I
    6. Electra2
  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.
  3. The second phase of this lab will be to define the precise reagent concentrations for your cell-free experiment. You will be assigned artwork wells with specific fluorescent proteins and receive an email with instructions this week (by April 24).