Week 11 Lab: Cloud Labs

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).

The amino acid sequences are shown in the HTGAA Cell-Free Benchling folder.

sfGFP: primary advantage is robust folding kinetics; it is engineered to fold correctly even when fused to insoluble proteins, making it highly resistant to aggregation in the crowded environment of a cell-free extract.

mRFP1: characterized by slow maturation kinetics and a tendency for photobleaching; the delay between peptide synthesis and chromophore formation can lead to an underestimation of protein yield in short-term reactions.

mKO2: features fast maturation and oxygen dependence; while it reaches peak fluorescence quickly, the final oxidative step of chromophore formation requires sufficient O2 levels, which may become limiting in deep-well plates.

mTurquoise2: known for high quantum yield and acid stability; its low pKa makes it less sensitive to the $pH$ drops that naturally occur as metabolic byproducts (like organic acids) accumulate during long-term cell-free incubation.

mScarlet_I: a high-brightness variant with accelerated maturation compared to earlier red FPs; however, it remains sensitive to the oxidative environment, as oxygen is required to complete the cyclization of its chromophore.

Electra2: optimized for ultra-fast maturation; its rapid “time-to-bright” makes it the ideal candidate for real-time monitoring of transcription-translation (TX-TL) kinetics where immediate feedback is 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.

Protein: mScarlet_I

Reagent Adjustment: Increase Glucose and Nicotinamide concentrations while utilizing a semi-permeable reaction seal.

Expected Effect:In a 36-hour run, the primary bottleneck for a bright red FP like mScarlet_I is the depletion of energy and the requirement for oxygen for chromophore maturation. By increasing Glucose and Nicotinamide, we extend the metabolic “runway” for $ATP$ regeneration via the NMP-Ribose-Glucose pathway; combining this with a semi-permeable seal ensures a constant influx of O2 to drive the oxidative maturation of the chromophore, thereby maximizing the total fluorescent signal over the extended incubation period.

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 4/24). Make sure that your final project slide is in the slide deck below to be included!

The final phase of this lab will be analyzing the fluorescence data we collect to determine whether we can draw any conclusions about favorable reagent compositions for our fluorescent proteins. This will be due a week after the data is returned (TBD!).