Week 3 HW: Cell-Free Protocols

AI assistance disclosure: I used an AI assistant (Claude) for brainstorming and feedback on this assignment, including ideas for how to approach the composition design and help with formatting this page. The written answers below are my own.

Note on this submission: Note on this submission: I have an exam on Thursday and ran out of time this week. The three compositions are first pass submissions, not optimized designs, and some of the questions are only partly answered or left for next week. I’d like to keep working on this course, so I’ll replace the placeholders with properly calculated compositions and complete the remaining answers by next week.

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 and should reflect the distinct expression and folding requirements of that protein.

A. Compare cell-free reaction compositions

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

When I saw this question, it sounded eerily similar to how Ca2+ is highly regulated in cells while K+ is much more abundant, because it helps maintain cellular homeostasis. My guess was that Mg2+ and K+ in the reaction would work the same way, and after searching the literature, it confirmed my hypothesis. In E. coli, K+ is present at high concentrations and plays a role in osmoregulation, whereas Mg2+ is kept at much lower concentrations and mainly plays a role in ribosome and nucleic acid stability. (I still need to fill in the exact concentration ranges.) (I think the concentration ranges matter a lot here, and it raises a question for me: should a cell-free reaction mimic the concentrations of a living cell, or go beyond what a cell would tolerate to maximize protein output? I also think this connects to the next question. My guess is that PEP+NTP reactions may need tighter tuning of Mg2+ than Ribose+NMP reactions, but this is just a hypothesis and I’d want to check it.)

Not only that, in a paper by Shin and Noireaux (J Biol Eng 4, 8, 2010; https://doi.org/10.1186/1754-1611-4-8), they said that Mg has to be tightly regulated for optimal protein synthesis. They also showed that Mg2+ and K+ need to be optimized for the specific plasmid you use.

So if I had to pick which one is more sensitive, I would guess Mg2+, since it’s the lower-concentration ion that matters so much for the ribosome.

That made me learn that I also have to calibrate my concentrations to the plasmid I use, instead of just copying them from another composition.

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

PEP+NTP is faster but more expensive. PEP is a direct energy source and the NTPs are ready-made building blocks for transcription, so the reaction doesn’t have to do much work first. Ribose+NMP is much cheaper, but the lysate has to convert the ribose and NMPs into ATP and NTPs on its own, which introduces more variables (and probably more variability between lysates).

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

Note: A recommended starting point would be to review at least one published formulation, such as PANOx-SP (Jewett & Swartz 2004), a cost-optimized formulation from Olsen et al. (2026), and/or an AI-designed formulation from Smith et al. (2026).

B. Design one reaction composition for each of the three proteins

Sign in to coopetition.htgaa.org and publish your first unique 20 µL CFPS reaction composition for each of the following (you can resubmit later): sfGFP, PETase, Reteplase.

sfGFP

PETase

Reteplase

Community Reagents

C. Asimov Kernel (optional)

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

Bonus: 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).