Week 3 HW: Cell-Free Protocols

Part A — Understanding the CFPS Formulation

  1. Why are potassium glutamate and magnesium glutamate used at different concentrations? Which is more sensitive? Potassium glutamate and magnesium glutamate are both salts, but they have different jobs in a cell-free protein synthesis reaction. Potassium glutamate is used at a much higher concentration to help create an ionic environment that is similar to the inside of an E. coli cell. Magnesium is needed at a much lower concentration because it directly affects important parts of protein synthesis, including ribosomes and nucleotides. Because of this, magnesium is the one I would expect to be more sensitive to small concentration changes. The PANOx-SP work also re-optimized magnesium when the energy system and reaction environment were changed, which helped me understand why it is treated as an important variable.
  2. What is the difference between a PEP + NTP system and a Ribose + NMP system? A PEP + NTP formulation supplies the reaction with nucleoside triphosphates such as ATP, GTP, CTP, and UTP directly, while phosphoenolpyruvate (PEP) helps provide energy for the reaction. This is a more established approach, but some of these reagents can be expensive. A Ribose + NMP approach starts with cheaper or less energy-rich building blocks and depends more on the metabolism that is still active in the cell lysate to build or regenerate the molecules needed for transcription and translation. Olsen et al. (2026) showed that an optimized low-cost cell-free formulation could even produce NTPs from nitrogenous bases and ribose. The main reason to explore this type of formulation is to lower the cost while still producing useful amounts of protein.
  3. Comparison of two literature CFPS compositions I compared the PANOx-SP/Cytomim approach described by Jewett and Swartz (2004) with the newer low-cost formulation developed by Olsen et al. (2026). The older system uses a fairly large set of added reagents, including potassium, magnesium, and ammonium glutamate; amino acids; ATP, GTP, CTP, and UTP; PEP; NAD; CoA; spermidine; putrescine; folinic acid; and other components. Published versions of this approach commonly use roughly 130–175 mM potassium glutamate, around 8–12 mM magnesium glutamate, about 10 mM ammonium glutamate, and around 30–33 mM PEP, although exact recipes can vary. Olsen et al. (2026) instead screened 1,231 different reagent formulations and developed an optimized 12-component formulation. Their goal was not only high protein production, but also lowering the number and cost of reagents. They reported about 2.4 g/L protein at the 15 µL scale and about 3.7 g/L at the 4 mL scale with oxygen supplementation, with an average estimated reagent-cost reduction of about 95% compared with previous formulations. To me, the main difference is that PANOx-SP is a useful established starting point, while the newer work shows that some of the traditional ingredients can be simplified or replaced when cost is part of the optimization goal. Part B — My Reaction Compositions For the designer portion, I started from the provided PEP reference recipes instead of trying to create completely new reactions from scratch. I changed one main salt variable for each protein so that the result would be easier to interpret. The three proteins also have different folding needs, so I kept the provided redox/folding components when they were important. Protein Reference Main change Reasoning sfGFP sfGFP PEP K(Glu) lowered from about 280 mM to about 250 mM sfGFP is the simplest target and does not require disulfide-bond folding. I used a moderate potassium-glutamate change as a simple one-variable test of the ionic environment. PETase PETase PEP Mg(Glu) increased from about 8.2 mM to 10.1 mM Magnesium affects the translation machinery and was one of the salt variables highlighted in the plate design. I kept DsbC and the existing redox components because this PETase has one disulfide bond. Reteplase Reteplase PEP K(Glu) increased from 200 mM to the designer maximum of about 224 mM Reteplase is the hardest target because it has nine disulfide bonds. I changed a salt variable but left the oxidizing system, GSSG/GSH, and DsbC alone so I was not also changing the folding conditions.

Submitted-design notes sfGFP: I lowered potassium glutamate while keeping the rest of the PEP reference formulation as constant as possible. This gives a straightforward test of whether a somewhat lower salt concentration changes folded sfGFP production. PETase: I increased magnesium glutamate to approximately 10.1 mM. PETase activity is measured using p-nitrophenol release rather than as a direct protein yield, so the useful comparison will be whether the new formulation increases enzyme activity. Reteplase: I increased potassium glutamate to approximately 224 mM, which was the maximum the designer allowed. I did not remove GSSG, GSH, or DsbC because Reteplase contains nine disulfide bonds and needs an oxidizing/folding-support formulation. Its readout is also enzyme activity rather than a direct physical protein yield. Community Reagent For the community-reagent portion, I would upvote FkpA, an E. coli folding chaperone that was already suggested by another student. I chose it because the difficult part of producing Reteplase is not just making the protein chain, but getting a protein with many disulfide bonds to fold into an active form. The community suggestion describes FkpA as a possible additional folding support alongside DsbC. Since this idea is already on the class page, upvoting it also avoids duplicating an existing suggestion. AI Use I used ChatGPT (OpenAI, GPT-5.6 Sol) while working through this homework. I used it mainly to help me understand what the Cell-Free Reaction Designer was showing, compare the reference formulations, choose simple one-variable changes for the three proteins, check that I had selected the correct protein template before submitting, and organize my notes into this write-up. I checked the final reaction settings in the class designer before submitting them. Examples of prompts/questions I used: • “k glu maxes out at 224” References 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., et al. (2026). Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature Communications, 17, 3478. https://doi.org/10.1038/s41467-026-69605-8