01
Energy & ATP regeneration
Keeps ATP and GTP topped up so translation doesn’t stall — usually the first thing to run out.
Cell-Free Synthesis Coopetition for Humans & AI
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Cell-Free Reaction Designer
Reagent concentrations in mM (proteins in mg/mL, enzymes in U/mL, DNA in ng/µL).
| Reference Plates | |||||||||
|---|---|---|---|---|---|---|---|---|---|
Mix 1 | sfGFP PEP 2026 18/18 | sfGFP Ribose 2026 13/13 | PETase PEP 2026 19/19 | PETase Ribose 2026 14/14 | Reteplase PEP 2026 21/21 | Reteplase Ribose 2026 21/21 | |||
| yield / activity | — | 0.61g/L | 0.46g/L | 314.6µM† | 274.8µM† | 10.9µM† | 0.81µM† | ||
| Salts | K(Glu) 875 mM | 313 | 280 | 345 | 200 | 300 | 200 | 273 | |
Mg(Glu)2 500 mM | 6.97 | 8.22 | 8.22 | 10.1 | 8.22 | 8.22 | 8.85 | ||
NH4(Glu) 1000 mM | — | 10 | 10 | 10 | |||||
Ammonium acetate ○ | — | ||||||||
| Amino acids | Amino acids 50 mM | 4.06 | 2 | 5 | 2 | 5 | 2 | 4.81 | |
Tyrosine 50 mM | 4.06 | 1 | 1 | 1 | 1 | 1 | 1.19 | ||
Cysteine 200 mM | 4 | 1 | 1 | 1 | 1 | 1 | 5 | ||
| Buffers | HEPES pH 7.5 1000 mM | 45 | 57.5 | 75 | 57.5 | 75 | 57.5 | 67.5 | |
Bis-Tris ○ | — | ||||||||
Phosphate (di:mono) 500 mM | — | 7.5 | |||||||
Phosphate (mono:di) 500 mM | — | 15 | 15 | 7.5 | |||||
K₂HPO₄ (dibasic) 500 mM | 5.63 | ||||||||
KH₂PO₄ (monobasic) 500 mM | 5.63 | ||||||||
| Energy substrates | Glucose 1110.15 mM | 6.94 | |||||||
Sodium pyruvate 908.76 mM | — | 9.09 | |||||||
Maltose 138.77 mM | — | ||||||||
Maltodextrin 300 mg/mL | — | ||||||||
Ribose 666.09 mM | 77.4 | 50 | 50 | 40 | |||||
PEP 100 mM | — | 30 | 30 | 30 | |||||
3-PGA ○ | — | ||||||||
— | |||||||||
Sodium hexametaphosphate 150 mM | — | ||||||||
Potassium formate 500 mM | — | ||||||||
| Cofactors | Folinic acid 10 mg/mL | — | 0.025 | 0.025 | 0.025 | ||||
tRNA ○ | — | ||||||||
CoA 50 mM | — | 0.25 | 0.25 | 0.25 | |||||
NAD 100 mM | — | 0.375 | 0.375 | 0.375 | |||||
cAMP 200 mM | — | ||||||||
Nicotinamide 100 mM | 3.13 | 4 | 4 | 4 | |||||
| Polyamines / redox | Putrescine 500 mM | — | 1.25 | 1.25 | 1.25 | ||||
Spermidine 250 mM | — | 1.56 | 1.56 | 1.56 | 1.25 | ||||
Dithiothreitol ○ | — | ||||||||
GSSG 170 mM | — | 4.04 | 4.04 | ||||||
GSH 100 mM | — | 1 | 1 | ||||||
Oxalic acid 500 mM | — | 3.75 | 3.75 | 3.75 | |||||
| Crowd | PEG-8000 10 % w/v | — | |||||||
| Additives | Brij-35 10 % v/v | — | |||||||
DMSO 60 % v/v | — | ||||||||
| TCA | Oxaloacetic acid 500 mM | — | |||||||
Succinic acid 500 mM | — | ||||||||
| Nucleosides / bases | Adenosine 25 mM | — | 0.406 | ||||||
Cytidine 25 mM | — | ||||||||
Guanosine 25 mM | — | ||||||||
Uridine 25 mM | — | ||||||||
Thymidine 25 mM | — | ||||||||
Adenine 25 mM | — | ||||||||
Guanine 25 mM | 0.156 | ||||||||
Cytosine 25 mM | — | ||||||||
Uracil 25 mM | — | ||||||||
Thymine 25 mM | — | ||||||||
| Enzymes | Catalase 50000 U/mL | — | 188 | ||||||
DsbC 100 µM | — | 5 | 5 | 5 | 5 | ||||
Pyruvate oxidase 10 mg/mL | — | ||||||||
| NTPs | ATP 100 mM | — | 1.25 | 1.25 | 1.25 | ||||
CTP 100 mM | — | 0.875 | 0.875 | 0.875 | |||||
GTP 100 mM | — | 0.875 | 0.875 | 0.875 | |||||
UTP 100 mM | — | 0.875 | 0.875 | 0.875 | |||||
| NMPs | AMP 100 mM | 0.625 | 3 | 3 | 0.5 | ||||
CMP 100 mM | 0.375 | 2.13 | 2.13 | 1 | |||||
GMP 100 mM | — | 2.13 | 2.13 | 1 | |||||
UMP 100 mM | 0.375 | 2.13 | 2.13 | 1 | |||||
| Water | Water | 1.27 | 0.47 µL | 0.17 µL | 1.23 µL | 0.23 µL | 0.63 µL | 0.65 µL | |
concentration rank
1. Potassium Glutamate 312.66 mM
2. HEPES-KOH pH 7.5 45.00 mM
3. Magnesium Glutamate 6.97 mM
4. Potassium phosphate dibasic 5.63 mM
5. Potassium phosphate monobasic 5.63 mM
6. 17 Amino Acid Mix 4.06 mM
7. Tyrosine pH 12 4.06 mM
8. Cysteine 4.00 mM
9. Nicotinamide 3.13 mM
10. AMP 625.00 uM
11. CMP 375.00 uM
12. UMP 375.00 uM
13. Guanine 156.25 uM
14. Ribose 11.625 g/L
15. Glucose 1.250 g/L
16. Water 1.275 uL
reagent supplement JSON
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Where to Start
The main considerations in cell-free protein synthesis — good starting points for your first hypothesis.
levers of cell-free synthesis · pick a layer to focus on
A cell-free reaction is a stack of layers, from making ATP to folding the final protein. Most good experiments change one layer at a time — choose the one you have a hypothesis about, start from a reference recipe, and test a few levels.
02
Transcription (DNA → mRNA)
How much mRNA T7 RNA polymerase makes from your template, and how long it lasts.
03
Translation (mRNA → protein)
Ribosomes reading the mRNA — usually the step that sets yield once energy is solved.
04
Ionic environment & pH
The salts and buffer every enzyme in the lysate works in.
05
Redox & disulfide bonds
Whether cysteines pair into the right disulfide bonds — essential for proteins that need them.
06
Folding, solubility & crowding
Getting correctly folded, soluble, active protein — not just a lot of polypeptide.
07
Cofactors & lysate metabolism
The small molecules the lysate’s own enzymes need to keep running.
08
Exploratory additives
Reagents on the list with less established roles in CFPS — good candidates for hypothesis-driven, single-variable tests.
09
Experimental strategy
How to learn the most from a limited number of wells.
Per research team, per round
384-well plates
4 plates
1,280 reactions
320 unique
96-well plates
1 plate
80 reactions
20 unique
2 mL tubes
20 tubes
20 reactions
5 unique
Protein Details
What we're expressing — structure, sequence, and how each target is measured.
target proteins
sfGFP superfolder GFP
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.
Length240 AA MW27,015 Da Disulfide bonds0 (2 free Cys) — standard CFPS conditions ChromophoreThr65-Tyr66-Gly67 (S65T), autocatalytic (requires O₂) QuantificationDirect fluorescence
MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGTRDHMVLHEYVNAAGITWSHPQFEK
PETase TfCut2 cutinase
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 AA MW33,157 Da Disulfide bonds1 (Cys274–Cys292) — standard CFPS conditions Active siteSer-His-Asp catalytic triad (α/β hydrolase) Quantificationp-nitrophenol release at OD 405 nm
MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFGGSGVSGWRLFKKIS
Reteplase vtPA serine protease
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.
Length399 AA MW44,136 Da Disulfide bonds9 (3 kringle-2 + 5 protease + 1 inter-domain) — requires oxidizing CFPS (DSB lysate + IAM) Active siteSer-His-Asp catalytic triad (serine protease) QuantificationAMC release from IPR-AMC peptide
MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRPGGSGVSGWRLFKKIS
assay setup
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 amount of product released (pNP for PETase, AMC for reteplase), read as an endpoint concentration in µM against each assay's product standard curve (equivalently, the initial release rate × read time). That is an observed activity measurement, not a verified physical yield, and the numbers are never comparable across the three targets.
sfGFP endpoint fluorescence
Key metricTiter — g/L folded protein (real yield) PlateCFPS reaction plate (direct) DilutionNone — read in-plate Volume20 µL
PETase OD 405 nm, kinetic
Key metricActivity — pNP released (µM endpoint vs standard curve), not a yield Dilution600× (final read) BufferPBS pH 8 Reads15 cycles, 0–28 min (~2 min apart) Rate windowFirst 6 cycles (0–10 min), linear phase
Reteplase 345 ex / 445 em, kinetic
Key metricActivity — AMC released (µM endpoint vs standard curve), not a yield Dilution2× (direct in CFPS plate) BufferAssay buffer e12461 — 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20 Reads15 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 reads Kinetic control reads per lysate batch — catalyst-agent-skills overview · joined to autonomous-cfps reference_results recipes
sfGFP RFU
24 49,930
endpoint · single read 312 loadable wells →
PETase OD 405
0.21 2.67
time = 0.00 h
15 reads · 0.47 h 312 loadable wells →
Reteplase RFU
2,593 53,149
time = 0.00 h
15 reads · 7.65 h 312 loadable wells →
standard curves assay calibration references
starting data reagent ranges across the plates we ran
Plate Designs — Reagent Ranges
Control runs — autonomous-cfps reference_results · sfGFP · PETase · Reteplase (OFAT rev3, Round 2)
sfGFPPETaseReteplase
Folding Simulation
From a flat chain of amino acids to the working shape each assay measures — scrub the timeline to watch secondary structure, hydrophobic collapse, and the disulfide bonds snap into place.
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The ribosome reads the mRNA codon by codon and links amino acids into one long chain, N-terminus to C-terminus. Right now it is only a sequence — a “string of beads” — with no 3-D shape.
sfGFP
240 aa — a superfolder GFP variant engineered to fold fast and robustly.
PETase
309 aa cutinase (TfCut2) — one chain, one domain.
Reteplase
~399 aa thrombolytic — a truncated t-PA (kringle-2 + serine-protease domains).
A stylised view of the folding hierarchy, not a molecular-dynamics run: the native shape uses each structure's real PDB coordinates (2B3P · 4CG1 · 1RTF) and the disulfide staples use the real SSBOND connectivity, while the extended “string” start and the morph between them are schematic so each step stays readable. Toggle X-ray to see every residue as a bead coloured by amino-acid chemistry — cysteines in gold — with the bonded pairs labelled by residue number (e.g. C241–C259). Drag a fold to rotate it.
Process Details
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.
sfGFP
PETase
Reteplase