Week 3 HW: Cell-Free Reaction Design
Zia Baig | HTBAA 2026 | October 6, 2026
A. Comparing reaction components
Potassium glutamate helps maintain the ionic conditions needed for protein synthesis. Magnesium glutamate supports ribosomes and many enzymes, but nucleotides and phosphate bind magnesium and change how much remains available. Magnesium is therefore the more sensitive variable: small changes can sharply reduce expression, while potassium usually has a broader useful range (Olsen et al., 2026).
A PEP/NTP formulation supplies nucleotide triphosphates for transcription and uses PEP to regenerate ATP. It is well established, but its inputs are costly and phosphate can accumulate. A ribose/NMP formulation relies on the lysate to generate ATP and convert monophosphates into triphosphates. This can lower reagent costs, but performance depends more on lysate metabolism and oxygen transfer (Olsen et al., 2026).
| Feature | PANOx SP (Jewett & Swartz, 2004) | RFopt (Olsen et al., 2026) |
|---|---|---|
| Energy / RNA | PEP 30 mM; ATP 1.25 mM; other NTPs 0.875 mM each | Ribose 50 mM; AMP 3 mM; other NMPs 2.15 mM each |
| K / Mg glutamate | 130 / ~8 mM | 362 / ~8 mM |
| Amino acids | 2 mM each | 5 mM each |
| Other inputs | Ammonium glutamate, oxalate, polyamines, NAD, CoA, folinic acid | HEPES 75 mM; phosphate 15 mM; glucose 10 mM; nicotinamide 4 mM |
| Main tradeoff | Established PEP system with costly inputs | Lower reagent cost, but greater dependence on lysate metabolism and oxygen |
B. Proposed 20 µL reactions
The table shows one reaction for each target. All volumes are in nL, in the designer’s 25 nL increments, and each reaction totals 20,000 nL. Each uses the matching DNA template. The fixed buffer contributes 200 mM potassium glutamate, 2.6 mM magnesium glutamate, 30 mM HEPES, and 1 mM each of the amino acid mix, tyrosine, and cysteine. All listed reagents were in stock on October 6, 2026 (Ginkgo Bioworks, 2026; HTBAA, 2026).
| Reagent (nL) | sfGFP | PETase | Reteplase |
|---|---|---|---|
| Base buffer | 2,000 | 2,000 | 2,000 |
| Lysate | 5,000 | 5,000 | 5,000 |
| Target DNA template | 2,000 | 2,000 | 2,000 |
| 17 amino acid mix | 1,600 | 400 | 400 |
| Ammonium glutamate | — | 200 | 200 |
| AMP | 600 | — | — |
| CMP / GMP / UMP | 425 each | — | — |
| ATP | — | 250 | 250 |
| CTP / GTP / UTP | — | 175 each | 175 each |
| Coenzyme A | — | 100 | 100 |
| DsbC | — | 500 | 800 |
| Folinic acid | — | 50 | 50 |
| HEPES KOH pH 7.5 | 900 | 550 | 750 |
| Magnesium glutamate | 225 | 300 | 225 |
| NAD | — | 75 | 75 |
| Nicotinamide | 800 | — | — |
| Oxidized glutathione GSSG | — | — | 475 |
| Reduced glutathione GSH | — | — | 200 |
| PEP | — | 6,000 | 6,000 |
| Potassium glutamate | 3,325 | — | — |
| Potassium oxalate | — | 150 | 150 |
| Potassium phosphate mix | 600 | — | — |
| Putrescine | — | 50 | 50 |
| Ribose | 1,500 | — | — |
| Spermidine | 50 | 125 | 125 |
| Nuclease free water | 125 | 1,725 | 625 |
| Total | 20,000 | 20,000 | 20,000 |
sfGFP. I would add 0.625 mM spermidine to the site’s ribose/NMP reference recipe and reduce the water by 50 nL. This changes one ingredient, so I could compare sfGFP fluorescence and cost per gram with the original recipe (Ginkgo Bioworks, 2026; Olsen et al., 2026).
PETase. I would use standard lysate and reduce DsbC from 5 to 2.5 µM, replacing the removed 500 nL with water. At the listed stock price, this saves $2.816 per reaction. I would check whether PETase activity remains high enough to improve activity per dollar (Ginkgo Bioworks, 2026; HTBAA, 2026).
Reteplase. I would keep the oxidizing DSB lysate and GSSG/GSH mixture, raise HEPES from 57.5 to 67.5 mM, and reduce DsbC from 5 to 4 µM. The lower DsbC amount saves about $1.126 per reaction. I would use IPR-AMC cleavage to test whether the reaction maintains activity (Ginkgo Bioworks, 2026; HTBAA, 2026).
I would compare each design with its unchanged reference and a no-DNA control, then repeat any promising result. The PETase and Reteplase assays report activity, not protein mass, so they cannot establish cost per gram on their own (HTBAA, 2026).
Community reagent: FkpA
FkpA is my choice because it may help fold disulfide-rich Reteplase. In an E. coli cell-free system, FkpA and DsbC improved antibody folding together (Groff et al., 2014). Its effect on Reteplase activity and cost remains to be measured.
References
Ginkgo Bioworks. (2026). CFPS coopetition overview and guidelines [Project specification]. https://docs.google.com/document/d/1E9RKXezEU5pMO5A10kkUHjHrfL5f8segL0QeNHXAs_E/edit
Groff, D., Armstrong, S., Rivers, P. J., Zhang, J., Yang, J., Green, E., Rozzelle, J., Liang, S., Kittle, J. D., Steiner, A. R., Baliga, R., Thanos, C. D., Hallam, T. J., Sato, A. K., & Yam, A. Y. (2014). Engineering toward a bacterial ‘endoplasmic reticulum’ for the rapid expression of immunoglobulin proteins. mAbs, 6(3), 671–678. https://doi.org/10.4161/mabs.28172
HTBAA. (2026). Cell free synthesis coopetition for humans and AI [Reaction designer]. https://coopetition.htgaa.org/cfps
Jewett, M. C., & Swartz, J. R. (2004). Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnology and Bioengineering, 86(1), 19–26. https://doi.org/10.1002/bit.20026
Olsen, M. L., Copeland, C. E., Sundberg, C. A., Aw, R., Shaver, Z. M., Rao, G., Swartz, J. R., Karim, A. S., & Jewett, M. C. (2026). Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature Communications, 17, Article 3478. https://doi.org/10.1038/s41467-026-69605-8
OpenAI. (2026). Codex [Large language model]. https://openai.com/codex/