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Field-standard fluorescent reporter — folds fast and glows green on its own, so expression reads directly in-plate with no purification. An industry-standard benchmark.
A cutinase that hydrolyzes the ester bonds of PET plastic into its monomers — terephthalic acid and ethylene glycol — which can be re-polymerised into new plastic, closing the recycling loop. A model biocatalyst for enzymatic plastic recycling and a test of expressing a disulfide-bonded enzyme.
Length309 AAMW33,157 DaDisulfide bonds1 (Cys274–Cys292) — standard CFPS conditionsActive siteSer-His-Asp catalytic triad (α/β hydrolase)Quantificationp-nitrophenol release at OD 405 nm
A clot-busting thrombolytic — it activates plasminogen into plasmin, which dissolves the fibrin holding a clot together, so it's used to treat ischemic stroke, myocardial infarction, and other acute thrombotic conditions. The hardest target here (9 disulfide bonds), proving CFPS can make complex therapeutics.
Key metric. sfGFP reports a true expression titer — g/L of folded
protein, read directly by fluorescence against a purified-sfGFP curve. PETase and reteplase
report enzyme activity — the initial product-release rate (pNP for PETase, AMC for
reteplase), expressed as an apparent g/L via each assay's standard curve. That is an
observed activity measurement, not a verified physical yield, and g/L values are never
comparable across the three targets.
sfGFP endpoint fluorescence
485 nm ex510 nm emGFP
Blue excitation light (485 nm) drives the chromophore to emit green (510 nm) — read directly in-plate.
The esterase attacks the ester bond of p-nitrophenyl hexanoate (pNPH): a water molecule splits the substrate into hexanoic acid and para-nitrophenol. In the alkaline buffer (pH 8) the para-nitrophenol loses a proton — it deprotonates to the para-nitrophenolate anion, whose extended, delocalised electron system now absorbs violet and blue light. With those wavelengths removed, the solution transmits the rest and reads yellow to the eye — tracked as absorbance at OD 405 nm.
Key metricActivity — pNP-release rate (OD 405 slope) → apparent g/L, not a yieldDilution600× (final read)BufferPBS pH 8Reads15 cycles, 0–28 min (~2 min apart)Rate windowFirst 6 cycles (0–10 min), linear phase
Reteplase 345 ex / 445 em, kinetic
IPR–AMC445 nm emIPRAMC✂
Reteplase is a serine protease: it hydrolyses the amide bond just after the arginine of the D-Ile-Pro-Arg peptide, releasing free 7-amino-4-methylcoumarin (AMC). Tethered to the peptide the coumarin is held dark, but once its amino group is freed it donates electrons into the ring's conjugated system. Ultraviolet excitation at 345 nm now lifts those electrons; as they relax they emit blue light at 445 nm — and the rate at which that fluorescence climbs scales with protease activity.
Key metricActivity — AMC-release rate (fluorescence slope) → apparent g/L, not a yieldDilution2× (direct in CFPS plate)BufferAssay buffer e12461 — 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20Reads15 reads over ~8 h — ambient between reads (kinetic loop)Rate windowEarly linear reads, OLS slope
Assay Details & Starting Data
How the signal is captured — kinetic plate reads and the calibration curves behind each unit — plus the reagent ranges across the plates we actually ran.
plate readsKinetic control reads — catalyst-agent-skills overview · joined to autonomous-cfps reference_results recipes
sfGFPRFU⤢
2449,930
endpoint · single read312 loadable wells →
PETaseOD 405⤢
0.212.67
time = 0.00 h
15 reads · 0.47 h312 loadable wells →
ReteplaseRFU⤢
2,59353,149
time = 0.00 h
15 reads · 7.65 h312 loadable wells →
standard curvesassay calibration references
starting datareagent ranges across the plates we ran
How the reactions are built and run — the shared automation backbone and the floor it runs on.
process
Shared prep backbone — Echo hitpick → Bravo stamp → incubate — then a protein-specific readout tail (highlighted). Click any step for its transfer specs.
