Week 11 HW: Bioproduction and Cloud Labs

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

Artwork link

I contributed a pixel in the middle of the letter O in the word LOVE, but the pixel was later replaced.

What I did like was that there is a chance to contribute to the automation protocol design.

What I guess could be better is if we had access to the past year’s experiments (at least for the baseline concentrations) in order to estimate the variability, better plan their 3 wells, and see what changes worked for specific fluorescent proteins. Overall, I think for the problem presented as is (identify a fluorescent protein to work with and present experiments using just 3 wells, with no prior knowledge available), the nature of experiments each student proposes could reveal some interesting personality traits that characterise the HTGAA community; that would be interesting to collect and analyse this information involving those students who have a psychology background.

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

1. Components and their role

E. coli Lysate • BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) – The lysate is an unfractionated content of the cytoplasm, it supplies ribosomes, tRNAs, aminoacyl-tRNA synthetases, elongation and initiation factors, and the metabolic enzymes. T7 RNA polymerase is the most important enzyme in the reaction; it’s a bacteriophage polymerase but has been integrated into the E.coli strain because it’s faster than the native polymerase and does not require transcription machinery, which I guess is important for the transcription not to be a limiting factor.

Salts/Buffer • Potassium Glutamate – Potassium is the main cation cells are adapted to, and glutamate is E.coli’s major cytoplasmic anion modulating osmolarity, and so both are recreating the native ionic environment. Potassium is needed for ribosomes to fold and assemble (by neutralizing negative phosphate charges on rRNA and allowing ribosomal subunits to not repel) and for translation itself, but too high concentration inhibits translation (promotes dissociation of the ribosomal subunits and initiation complex destabilize) as many of the interactions involved in the process are reversible electrostatic; glutamate unlike chloride is large and it does not interact with protein surfaces with positively charged regions and functional interfaces. • HEPES-KOH pH 7.5 – It’s a buffer needed to keep pH for the optimal work of transcription and translation enzymes by counteracting accumulating acidic byproducts of energy metabolism. Buffer titration experiments may be beneficial. • Magnesium Glutamate – Magnesium is the major cofactor for T7 polymerase (uses two ions at the active site to form phosphodiester bonds), it defines structure and function of ribosomes (neutralizes negative phosphate charges of rRNA), it stabilises nucleotides and RNA (binds to phosphates of nucleotides, mRNA, tRNA); and glutamate is a safe (large) counter-ion maintaining osmolarity as in potassium glutamate. Magnesium is the most nonlinear parameter in the cell-free reaction system; at too low a concentration, transcription and translation efficiency decrease, and upon too high concentration, RNA molecules crosslink, and nucleotides precipitate as magnesium phosphate salts. • Potassium phosphate monobasic/dibasic – another buffer system, but for a different pH range than HEPES, and so may add the capacity to counteract pH drift from accumulating acetate. Phosphate serves as an energy metabolism substrate and regenerates ATP and nucleotide triphosphates, but too much phosphate can diminish magnesium (as the magnesium salt is insoluble) and inhibit the reaction.

Energy / Nucleotide System • Ribose – replenishes the NTP pool, in the system where nucleoside monophosphates and free guanine are supplied instead of ready-made triphosphates. It is converted to the sugar-phosphate backbone (phosphoribosyl pyrophosphate) that is needed to build nucleotides. • Glucose – primary energy source, substrate for glycolysis to generate ATP for transcription and translation. Acetate produced as a byproduct shifts the pH to acidic. • AMP, CMP, GMP, UMP – ribonucleoside monophosphates phosphorylated by kinases in the lysate, precursors to ATP, GTP, CTP, UTP for mRNA synthesis. • Guanine – converts to guanine nucleotides through the purine salvage pathway. Replenishes GTP pool that is consumed in transcription and translation.

Translation Mix (Amino Acids) • 17 Amino Acid Mix, Tyrosine, Cysteine – building blocks for proteins, fluorescent proteins in our case. Tyrosine is added separately because of its different solubility (in high pH), and cysteine oxidizes quickly. Additives • Nicotinamide – precursor for NAD⁺/NADH indispensable for glycolysis; these are not consumed in transcription or translation. Backfill • Nuclease Free Water – dissolves all the components, is deprived of RNases or DNases that would degrade the template and mRNA.

2. Describe the main differences between the 1-hour optimized PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix.

The 1-hr master mix is optimized for speed and contains triphosphates and a high-energy phosphate donor (PEP, to quickly recharge ATP) as well as NTPs to quickly supply transcription and translation, higher concentration HEPES as the only buffer, and a number of additives including energy equivalents and redox cofactors, and stabilizers for nucleic acids.

The 20-hour mix is optimized to sustain the long reaction and for the cost. It contains nucleoside monophosphates and free guanine phosphorylated by lysate enzymes, ribose and glucose a phosphate buffer, in addition to HEPES, but also as a substrate for energy-regeneration to recharge nucleotides, higher concentrations of amino acids as more total protein is synthesized through a longer reaction, and only nicotinamide as an additive needed as a precursor for regenerating NAD.

3. How can transcription occur if GMP is not included but Guanine is?

Guanine converts to guanine nucleotides through purine salvage pathway. Replenishes GTP pool that is consumed in transcription and translation.

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):

sfGFP – is optimized for efficient folding (a very small fraction of molecules fail to fold) and fast maturation (in minutes), making sfGFP the most reliable reporter. It’s also very stable as its barrel is quite resistant to proteases in E.Coli lysate, and so the fluorescent signal mostly accumulates.

mRFP1 – slow chromophore maturation (~ tens of minutes, closer to 1 hr) and relatively low brightness (lower than that of mScarlet-I), so no fast kinetics can be observed. More synthesized protein would help to increase brightness. It’s historically interesting as mRFP1 opened the palette for monomer red and orange fluorescent proteins; mRFP1 was developed with a large number of mutations, and in its slow maturation, the fluorophore passes a stage with green fluorescence.

mKO2 - maturating faster than its parent mKO, but with pH of 5.5, which can be reached with accumulating acetate if HEPES is not buffering it, half of the molecules lose fluorescence. The latter feature was actually part of the mKO2 design that was developed for cell cycle monitoring timer/indicator in the Fucci system, where green and red proteins are fluorescent in different cell cycle phases; the name of the protein is actually Kusabira-Orange, which is probably the name of the coral, and “Kusabira” means mushroom in old Japanese, as the coral reminded one of a mushroom.

mTurquoise2 – very high resistance to acid, fast maturation, and high quantum yield for a cyan protein. Its pKA is much lower than that of mKO2 (3.1 vs 5.5), which can be useful in reactions where pH is predicted to shift to acidic; however, the dynamic range is narrow because of the high autofluorescence of lysate components, having excitation and emission overlapping with that of mTurquoise2.

mScarlet_I – matures faster than mRFP and mScarlet (~40 min vs ~100 min of mScarlet) and has slightly lower brightness than mScarlet. It can capture fast dynamics, and its pKa is ~5.4 (moderate acid resistance).

Electra2 – it’s a blue fluorescent protein engineered from mRuby3, and it can form aggregates, which affects the reproducibility of the signal in cell-free systems. Also, the dynamic range is narrow as well (lower than that of green and red reporters) for the same reason of high autofluorescence in near UV.

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.

Higher concentration of HEPES buffer may make the reaction more resistant to pH drift due to acetate accumulation through the long reaction and protect sfGFP signal (decrease chromophore protonation). Some HEPEs concentration optimum (a plateau in fluorescent signal) is expected upon increasing HEPES concentration because the ionic strength also increases.

Additionally, sfGFP expression and green fluorescence could be increased by adding magnesium (but the effect is not specific to sfGFP), and reagents modulating oxygen availability could improve maturation and fluorescence of sfGFP over the long incubation time.

3. Define the precise reagent concentrations for a cell-free experiment.

8 wells were registered with the following parameters:

(Link for the wells and reagents)