Week 11 HW: Bioproduction & Cloud Labs

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

I’m so sorry, I didn’t meet the deadline to submit but I will definitely be applying to be a TA this fall. :)

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.

BL21 (DE3) Star Lysate (includes T7 RNA Polymerase): E-Coli cells that have been opened, where their inside machinery provides the mechanisms for transcription and translation of your proteins. The BL21 has a modification to help your protein not get degraded, the DE3 carries an inserted polymerase gene for fast transcription from t7 promoters, and Star has a modification so that the mRNA isn’t chewed up before translation.

Potassium Glutamate: Provides K+ which is integral for ribosome assembly, tRNA folding and translation fidelity. Glutamate specifically gives K+ without the downsides of KCI.

HEPES-KOH pH 7.5: A sort of pH sponge that soaks up the acidic waste produced during the reaction that burns sugar for energy. This helps preserve enzymes and ribosome activity by holding the pH near 7.5 (optimal operating point mimicking E.Coli’s native pH).

Magnesium Glutamate: Magnesium is an integral concentration-sensitive component in cell-free systems that allows for ribosome stabilization, codon pairing, and ATP reactions. Too little levels cause the ribosomes to dissociate, and too much causes translation to be error prone.

Potassium phosphate monobasic: Supplies inorganic phosphates that lysate kinases attach onto NMPs so that they can become NTPs for transcription and translation.

Potassium phosphate dibasic: Supplies the same inorganic phosphate pool as above that kinases use to charge NMPs into NTPs and ADP into ATP. It’s paired with the monobasic at 1.6:1 to help the reaction pH stable at 7.5 over long incubation.

Ribose: A sustainable and readily available form of carbon/sugar that the cell uses as raw material to build its nucleotides from scratch, and that gets burned through during ATP generation. This slow release of sugar and salvage strategy was the core innovation that lets the reaction run for 20 hours.

Glucose: Another cheap sugar that feeds into glycolysis to generate ATP. It complements ribose by providing a steady metabolic fuel to the lysate, and allowing ribose to be freely used for building nucleotides.

AMP: cheap, low-energy form of adenosine.The lysate’s kinases boost it up the energy ladder, allowing it to be used for transcription and translation.

CMP: cheap, low-energy form of cytidine. The lysate’s kinases boost it up the energy ladder, allowing it to be used for transcription and translation.

GMP: cheap, low-energy form of guanosine. It’s supplied as free guanine, letting the lysate enzymes build GMP from scratch.

UMP: cheap, low-energy form of uridine. The lysate’s kinases boost it up the energy ladder, allowing it to be used for transcription and translation.

Guanine: Free base form of G, the nitrogenous base with no sugar and no phosphate, a cheap precursor of G. The lysate’s salvage enzymes attach with other material to build GMP, which kinases then up to GTP for use in transcription.

17 Amino Acid Mix: A premixed solution of 17 of the standard amino acid, which are building blocks the lysate’s load onto tRNAs, and then ribosomes string together into proteins during translation. The other 3 amino acids are added separately because of solubility and stability issues that would otherwise cause them to degrade.

Tyrosine: One of the 20 amino acid building blocks added separately as it has poor solubility at neutral pH. Instead it is prepared as a separate stock dissolved in highly basic water. The small amount of alkalinity added with it is absorbed by the HEPES buffer mentioned earlier.

Cysteine: One of the 20 amino acid building blocks added separately because its free thiol group oxidizes rapidly in storage. It’s kept as a separate stock to ensure the cysteine in the reaction is usable when needed during translation.

Nicotinamide: Vitamin B3, which is the precursor that the lysate’s salvage enzymes use to build NAD+, an electron shuffling molecule that glycolysis needs to produce ATP. It’s topped up to replenish the NAD+/NADH pool as it gets continuously consumed over the 20 hour incubation. Backfill

Nuclease Free Water: Ultra-pure water that is treated prior to ensure it is free of RNases and Dnases (enzymes that can destroy RNA and DNA or contaminate the reactions).

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 comes down to supply: the 1-hour systems supplies are already pre-charged and ready to use NTPs (ATP, GTP, CTP, UTP) and PEP-Mono supplies a phosphate donor for fast ATP regeneration. This gives immediate but short-lived transcription and translation, burning out within an hour as the free phosphate accumulates magnesium. The 20-hour system supplies cheap precursor NMPs (AMP, CMP, UMP), free guanine, ribose and glucose, relying on the lysate to slowly build the pathways for NTPs and ATP regeneration. Paired up with the NAD+ replenishment, the system produces over a much longer incubation time, at a lower cost but also slower speed.

Bonus question: How can transcription occur if GMP is not included but Guanine is?

The lysate has a salvage enzyme called HGRPT that takes free guanine and attaches it to PRPP (an activated form of ribose) to produce GMP in a single enzymatic step. The lusate’s kinases then boost GMP up the energy ladder into GDP, and then GTP, which RNA polymerase then incorporates as the G letter during transcription. Therefore, GMP is not missing from the reaction, but is being built on demand from cheaper precursors.

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)

sfGFP: A very rapidly-maturing weak dimer with a maturation time of 13.6 minutes, allowing for the chromophore to reach its fluorescence state quickly enough to give a strong readout within the CFPS reaction timeframe. It was engineered to fold robustly even when fused to poorly-soluble partners, crucial in cell-free systems.

mRFP1: A slowly-maturing monomer with a maturation time of 60 minutes and a pKA of 4.5, so its chromophore needs significantly more oxidation and time to reach the red-emitting state. In a cell-free system reaction, a lot of the protein produced may still be immature non-fluorescence at readout, underestimating its actual yield. However low pKA means once mature the signal stays stable as the reaction acidifies.

mKO2: Has a maturation time of 108 minutes because of its three-ring chromophore requiring extra oxidative cyclization steps beyond standard chromophore formation. This makes it sensitive to oxygen-limited environments of a close cell-free system tube, where the chromophore maturation may stall or remain incomplete.

mTurquoise2: Strong quantum yield of 0.93 (93% of the absorbed photons are re-emitted as fluorescence) giving very bright readout per molecule even at modest CFPS yields. The kPA is very low at 3.1 making it strong against pH drift from glycolytic acid waste during long incubations, so signal stables stable even as the pH drops.

mScarlet_I: The link to this one gives mScarlet instead of mScarlet_I, so I typed in mScarlet_I in the search, and used that link’s info instead. It’s a rapidly maturing monomer at 36 minutes engineered from its parent’s (mScarlet) mutation. The quantum yield is still pretty solid. The faster maturation allows more synthesized proteins to reach fluorescence state within the finite oxygen timeframe, even if it glows a bit more dim than its parents.

Electra2: A monomeric blue fluorescence protein engineered from mRuby3, achieving a pretty high quantum yield of 0.76. The maturation time is not reported in the link provided.

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.

You can’t add oxygen to the master mix the same way you’d add nicotinamide. However, you can still engineer oxygen availability to be more readily available for mKO2, since it’s sensitive to oxygen-limited environments. It’s limited due to three main reasons:

Standard chromophore oxidation (every GFP-family FP requires molecular oxygen to oxidize its chromophore into its fluorescence state)
Extra thazoline ring (this requires an additional oxidative cyclization step)
Reaction-tube hypoxia (closed CFPS tubes go hypoxic as the lysate is burning sugar through glycolysis, eating the available O2.

Ways you can add more oxygen:

Use a larger headspace above the tube (run it in a tube where it’s mostly air-volume above the liquid)
Increase surface area, running it in a flat-bottom plate instead of a tube, allowing for better oxygenation.
Put it in a shaking incubator, allowing for more oxygen infusion. You can further improve on this by using breathable seals.
Reduce glucose, meaning less glycolysis and less oxygen consumption.
Add some sort of O2 carrying additives to extend oxygenation.

You could also hypothetically run the reaction in an O2 enriched chamber, or set up some sort of continuous-exchange CFPS with an oxygen permeable membrane. There are many ways you can supplement more oxygen into the reaction, allowing for more efficient mKO2.