Week 11 HW: Bioproduction & Cloud Labs
Part A: The 1,536 Pixel Artwork Canvas | Collective Artwork
(1) I didn’t manage to contribute to the artwork. (2) I liked the fact that every pixel removed / or placed directly influences a cell-free protein synthesis optimisation experiment, it makes the art feel alive and purposeful. A couple of suggestions and ideas I thought, instead of removing pixels to end the experiment, perhaps next year could feature a growth versus decay mechanic where different biological inputs (represented by different colours) compete for dominance on the plate. It would be interesting to have a secondary window showing a live feed or a time-lapse of the actual laboratory plate being manipulated by the cloud lab robots as we click, although the slider showing the bioart over time is incredible. To prevent griefing or to encourage rapid collaboration during peak hours, the cooldown could scale based on the complexity of the protein being synthesised in that specific quadrant.
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 - contains protein translation machinery - ribosomes, tRNAs, elongation factors, chaperones and here, T7 RNA Polymerase specifically included to read T7-promoter-driven DNA into mRNA. T7 RNAP is preferred because it is a single-subunit enzyme, and therefore is simpler and faster than the multi-subunit E. coli RNAP.
Salts/Buffer - regulate pH in the cell. Regulates ionic strength, pH (~7.5) and Mg²⁺ concentration important for ribosome assembly and polymerase activity.
Salts/Buffer (K-glutamate, HEPES-KOH, Mg-glutamate, K-phosphate) These maintain ionic strength, pH (~7.5), and Mg²⁺ concentration — all critical for ribosome assembly and polymerase activity. Glutamate is used as the counterion partly because it is a natural E. coli cytoplasmic osmolyte.
Energy / Nucleotide System - (Ribose/Glucose AMP, CMP, GMP, UMP, Guanine) - Nucleotides are substrates for transcription (NTPs built from NMPs + phosphate donors). Ribose and glucose feed metabolic pathways that regenerate energy carriers. This group is where ATP regeneration strategy lives.
Translation Mix (Amino Acids) - Amino acids are the single unit substrates for the ribosome. Tyrosine and cysteine are listed separately because of solubility issues at the stock concentration used for the other 17.
Nicotinamide - Acts as an NAD⁺ precursor, supporting redox reactions in the energy metabolism pathways that regenerate ATP.
Nuclease-Free Water - Backfill to final reaction volume, it ensures no RNase contamination that would degrade mRNA templates.
(2) 1 hr incubation has a PEP based system which has NTPs supplied and 20 hr incubation has a ribose based system (more sustainable, precursor driven and indirectly supplied NTPs).
(3) Bonus question: How can transcription occur if GMP is not included but Guanine is? It can because the cell-free extract contains enzymes that were freeze-dried and are reactivated by water. Those enzymes include the purine salvage pathway machinery to make transcription occur. Once water rehydrates the extract and those enzymes become active again, hypoxanthine-guanine phosphoribosyltransferase (HGPRT) can use the cosubstrate hypoxanthine-guanine phosphoribosyltransferase (HGPRT) requires 5-phosphoribosyl 1-pyrophosphate (PRPP) as its necessary co-substrate to attach guanine to the ribose-5-phosphate sugar, forming guanosine monophosphate (GMP).
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 - can fold correctly and fluoresce whilst being fused to poorly folded proteins or expressed in the complex chemical environments of cell-free extracts.
mRFP1 - quite slow maturation time compared to newer variants, causes a significant delay between protein synthesis and the appearance of a red signal in the painting.
mKO2 - high oxygen dependence for chromophore maturation, meaning that if the cell-free reaction has not got sufficient oxygen, the orange readout can be very diminished.
mTurquoise2 - valued for its high photostability and quantum yield, providing a very bright and steady cyan readout that resists bleaching during prolonged imaging or observation.
mScarlet_I - features rapid maturation making it one of the fastest red fluorescent proteins for real-time tracking of expression in cell-free reactions.
Electra2 - fast-maturing and bright green-yellow protein optimised for high-speed readout, often used in systems where rapid signal generation is critical to distinguish early expression dynamics.
(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.
To maximize the fluorescence of mRFP1 over 36 hrs of incubation, increasing the concentration of glucose and ribose sugars would address its slow maturation time.
Hypothesis By increasing the concentration of glucose and ribose, we could extend the metabolic lifespan of the cell-free reaction; this allows the reaction to remain energetically active long enough for the slow-maturing mRFP1 chromophore to fully transition into its fluorescent state before the system’s machinery degrades.
We would observe a significantly higher final fluorescence intensity for mRFP1 at the 36-hour mark, as the prolonged energy supply prevents the starvation of the reaction before the protein has had sufficient time to mature and glow.
(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). You can begin composing master mix compositions here.
(3)
