Week 3 HW: Cell-Free Reaction Design (Coopetition)

Week 3 Homework: Designing a Cell-Free Reaction Composition

Due: Tuesday, October 6, 2026

Coopetition website: coopetition.htgaa.org

Goal: Using the collective intelligence of the HTBAA community and accessible AI systems, design novel cell-free protein synthesis (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 and reflect the distinct expression and folding requirements of that protein.

A. Compare Cell-Free Reaction Compositions

A1. Potassium glutamate vs. magnesium glutamate

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

My answer:

Based on on this paper: https://pmc.ncbi.nlm.nih.gov/articles/PMC3161345/ the concentrations differ beacause each expierment requires a different level of patassium and magnesium to work - why would you assume it is static? magnesium is much more senitive

A2. PEP+NTP vs. Ribose+NMP

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

My answer:

Based on this paper: https://www.nature.com/articles/s41467-026-69605-8

Both the (1) PEP+NTP and the (2) Ribose+NMP are ways of providing the cell-free system enegry for the reaction. (1) contains pre-built energy and the pre-defined nucleotides so it is faster compared to (2) which contains the sugar that needs to be turned into engergy and the 1/2 built nucleotide strain that needs to be completed. I am not sure why anyone would use (2) over 1 since it is slower.. unless it is more costly? I don’t see that in the article.

A3. Compare two published compositions

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

Suggested starting points: PANOx-SP (Jewett & Swartz 2004), the cost-optimized formulation from Olsen et al. (2026), and/or the AI-designed formulation from Smith et al. (2026).

Composition 1:

This is a high-performance reaction - it uses PEP+NTP

Composition 2:

This is a low-cost reaction - it uses Ribose+NMP - which answers my question from the prior question :-)

Comparison of reagents and concentrations:

For energy ANOx‑SP uses PEP - high energy but expensive Olsen replaces PEP with Ribose - lower energy but cheaper

For RNA building blocks ANOx‑SP uses NTP - preassembled nucluotides Olsen uses NMP - sysetm turns into NTP - slower and cheaper

Other Factors (NAD, CoA, tRNA, etc.) ANOx‑SP includes all possible cofactors Olsen includes a lot less - paper argues most are not needed

Salts ANOx‑SP - PEP/NTP uses higher magnesium and potassium levels Olsen adjusts based on cheaper chemstry (I think?)

Amino Acids ANOx‑SP - standard 20‑amino‑acid mix at ~2 mM each Olson - less to lower costs

B. Design One Reaction Composition for Each of the Three Proteins

Rules: total reaction volume is 20 µL (20,000 nL). Fixed components (lysate 5,000 nL, DNA template 2,000 nL, base buffer 2,000 nL) take 9,000 nL. I choose up to 11,000 nL from the reagent menu; water fills the rest. All volumes must be multiples of 25 nL.

B1. Publish my first compositions on coopetition.htgaa.org

Question: Sign in to coopetition.htgaa.org and publish your first unique 20 µL CFPS reaction composition for sfGFP, PETase, and Reteplase. For each composition, specify the reagents you would adjust from the relevant categories, including salts, energy sources, building blocks, cofactors, and redox components.

sfGFP composition

My answer (reagents, volumes, and reasoning):

I took the default - just figuring out the user interface

PETase composition

My answer (reagents, volumes, and reasoning):

I took the default - just figuring out the user interface

Reteplase composition

My answer (reagents, volumes, and reasoning):

I took the default - just figuring out the user interface

B2. Community Reagents suggestion

Question: Contribute (or upvote) at least one reagent to the “Community Reagents” section of the website and provide a short rationale. Include how you decided on that specific reagent and provide links to any relevant research.

Reagent I suggested or upvoted:

FkpA - it was the first one listed

How I decided and my rationale:

FkpA - it was the first one listed

Links to relevant research:

N/A

B3. cfps-coopetition forum

Question: 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.

My answer (confirmation, plus my optimization idea if I posted one):

So the link just goes to

https://forum.htgaa.org/#narrow/channel/43-cfps-coopetition

which I joined and posted a test message

C. Asimov Kernel (Optional)

C1. Build T7-compatible plasmids

Question: Build T7-compatible plasmids for cell-free expression using the amino acid coding sequences provided for sfGFP, PETase, and Reteplase.

NA

Type your answer here.

C2. Bonus: T7 helper plasmid for Reteplase

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.).

My answer:

NA

Use of AI

If I used AI, I need to cite the AI client and the prompts I used, and be able to explain and justify my design reasoning in recitation.

AI tools used:

Claude (Anthropic), model Claude Opus 5.5, used through the Claude desktop app with access to my homework folder. I used it to summarize the assignment in plain language and to set up the structure of this homework page. It did not write my answers or design my reaction compositions.

Prompts used:

  1. To understand the assignment:

    review the two documents found here: ‘/Users/jamiedixon/Documents/MIT - HTBMAA/class 03’ tell me what I am supposed to be doing as if I am in 6th grade

  2. To set up this page:

    OK I created the _index page for week 3 here update the mark down to sections for my homework that I have to answer. Include the question and then leave a block where I can type in my answer

  3. To give Claude the location of the page:

    sorry, here is the file ‘/Users/jamiedixon/Documents/MIT - HTBMAA/webpages-2026b/content/week-03-HW-coopetition/_index.md’

  4. To document AI use:

    Fill in the AI tools used section and the prompts used so far

References

  • 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. doi:10.64898/2026.02.05.703998
  • 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