Week 3 HW: Cell-free Reaction Design

Full assignment context

3. ASSIGNMENT: CFPS REACTION DESIGN FOR SFGFP, PETASE, RETEPLASE

Goal: using the collective intelligence of the HTBAA community and accessible AI systems, design novel CFPS reaction compositions for each of the three target proteins: sfGFP, PETase, and Reteplase to reduce the cost per gram of functional protein. Each design should be compatible with the experimental framework described above and should reflect the distinct expression and folding requirements of that protein. You can learn more about the details of the coopetition here.

A. COMPARE CELL-FREE REACTION COMPOSITIONS (20 minutes)

Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?

Magnesium glutamate is necessary, and some of it is diverted in the experiments where NTPs, PEP, and phosphate buffers are present. In reactions where fewer phosphates are present, this is decreased to the necessary level given the amount that will be diverted. It is also more sensitive than potassium glutamate, going from concentration of 20mM to 24mM caused the yield to change 27% (and 10% in the other direction) in the PANOx-SP system, vs lower than 10% for K-glutamate. For the Cytomim system, a 2mM change in mM of Mg glutamate had a larger impact than a 70mM change in K glutamate on the upside, and a 2mM decrease in Mg glutamate was roughly equivalent to a 30mM decrease in K glutamate. (Table II Jewett & Swartz 2004)

Explain how a PEP+NTP reaction formulation is different from a Ribose+NMP reaction formulation.

PEP+NTP is the Jewett & Swartz approach. Translation burns ATP and GTP fast and PEP enables regeneration of ATP. This however causes accumulation of inorganic phosphate, which inhibits the reaction at about 30mM.

Ribose+NMP is Olsen’s approach that aims to solve this inefficiency. It consumes glucose and ribose to get glycolysis, with ribose also serving as the needed sugar. RNA polymerase can’t use monophosphates and in this case phosphate is consumed by lysate kinases so it doesn’t accumulate. It is a bit more circular in that the byproduct of the energy usage is used for something productive. Finally, this lower cost approach shows more sensitivity to pH..

Choose two cell-free reaction compositions from the literature, compare and contrast the choice of reagents and the concentration of reagents.

PANOx-SP (Jewett & Swartz 2004) and 1793122_35 (Smith 2026) had distinct approaches, mainly because the latter was trying to bring the overall cost down for the reaction. The original PANOx-SP reaction was optimizing for yield without considering cost at all. 1793122_35 kept the decreased Mg glutamate (8.8mM) that the Cytomim system started and other experiments continued, with increased K glutamate (273.2mM). It also however removed both Mg and K acetates. pH was balanced through HPES-KOH (67.5mM) and K phosphates (7.5mM di and mono). It used sodium pyruvate (9.1mM), glucose (8.3mM), and ribose (40.0mM) instead of PEP for energy, a more circular and self sustaining energy source. It also swapped ATP/CTP/GTP/UTP for AMP/CMP/GMP/UMP and increased amino acids (4.8mM).

B. DESIGN ONE REACTION COMPOSITION FOR EACH OF THE THREE PROTEINS (40 minutes)

Sign in to coopetition.htgaa.org and publish your first unique 20 µL CFPS reaction composition for each of the following (don’t worry, you can resubmit later):

sfGFP PETase Reteplase

Submission Link

For each composition, specify the reagents you would adjust from the relevant categories, including salts, energy sources, building blocks, cofactors, and redox components.

As a HTBAA research team, we will have the opportunity to suggest new reagents to add to the cell-free reagent inventory. Contribute (or upvote) at least one reagent to the “Community Reagents” section of the website and provide a short rationale. Include in your homework documentation how you decided on that specific reagent and provide links to any relevant research.

Visit the cfps-coopetition forum page to ensure you will be able to take part in the discussion. Bonus: send a note in the channel about a CFPS optimization idea that you have! If you would like clarification on aspects of the project, please message in this dedicated channel.

C. ASIMOV KERNAL (OPTIONAL)

Build T7 compatible plasmids for cell-free expression using the amino acid coding sequences below:

sfGFP MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEKGTRDHMVLHEYVNAAGITWSHPQFEK

PETase MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTGTEASIAWLGERIASHGFVVITIDTITTLDQPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVATHAKPFYNSLPSSISKAYLELDGATHFAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVEEYRSTCPFGGSGVSGWRLFKKIS

Reteplase MEKKIWSHPQFEKGGSGENLYFQSGGASPAGGGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMRPGGSGVSGWRLFKKIS

Bonus question: design a T7 helper plasmid that you could add to the cell-free reaction to improve the yield of functional reteplase (general chaperones, disulfide-bond machinery, etc).

  1. Use of AI This HW assignment began to hit the boundaries of my biology knowledge, and despite having a strong engineering foundation, I was having a hard time understanding the parts and constraints of this system. So I asked Claude to explain this system to me in a way that was less reliant on the biology knowlege, which helped a lot in understanding which pieces were functioning as energy, inhibitants, voltage regulators, etc.

I also did multiple iterations of trying to answer each question on my own and asking Claude to explain when I was misunderstanding something.

Finally, once I understood the system a bit better, I prompted Claude to come up with a few compositions given a focus on trying to manage pH in the lowest cost systems to get some ideas of which direction to go.

  1. REFERENCES AND RESOURCES
  • Smith, A. A., et al. (2026). Using a GPT-5-driven autonomous lab to optimize the cost and titer of cell-free protein synthesis (Ginkgo Bioworks × OpenAI). bioRxiv preprint. DOI: 10.64898/2026.02.05.703998. https://www.biorxiv.org/content/10.64898/2026.02.05.703998v1
  • 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. DOI: 10.1038/s41467-026-69605-8.
  • 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. DOI: 10.1002/bit.20026.
  • Silverman, A. D., Karim, A. S., & Jewett, M. C. (2020). Cell-free gene expression: an expanded repertoire of applications. Nature Reviews Genetics, 21, 151–170. DOI: 10.1038/s41576-019-0186-3.
  • Rasor, B. J., Vögeli, B., Landwehr, G. M., Bogart, J. W., Karim, A. S., & Jewett, M. C. (2021). Toward sustainable, cell-free biomanufacturing. Current Opinion in Biotechnology, 69, 136–144. DOI: 10.1016/j.copbio.2020.12.012.

Folder with pdfs: https://drive.google.com/drive/folders/1mWn6ncwFQg61ZbjsJU2XYCL8q5wGNJ3x?usp=sharing