Week 3 HW
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)
1. Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?
The concentrations of K-glutamate and Mg-glutamate differ between CFPS formulations because K⁺ and Mg²⁺ have distinct biochemical functions and their optimal concentrations depend on the composition of the entire reaction. K⁺ primarily establishes the ionic environment required for efficient translation, whereas Mg²⁺ directly interacts with ribosomes, nucleic acids, ATP and other metabolites. Changes in NTP, phosphate and other charged components alter Mg²⁺ availability, so the Mg²⁺ concentration must be adjusted accordingly. While the concentration of both matter, research has shown that Mg2+ has a relatively narrower optimum, compared to that of K+, with deviations strongly affecting protein synthesis (Banks et al., 2022).
References:
- Banks, A. M., Whitfield, C. J., Brown, S. R., Fulton, D. A., Goodchild, S. A., Grant, C., Love, J., Lendrem, D. W., Fieldsend, J. E., & Howard, T. P. (2022). Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions. Computational and Structural Biotechnology Journal, 20, 218 229. https://doi.org/10.1016/j.csbj.2021.12.013
2. Explain how a PEP+NTP reaction formulation is different from a Ribose+NMP reaction formulation.
PEP + NTP and ribose + NMP formulations differ mainly in how they provide energy and nucleotides for protein synthesis. In a PEP + NTP system, PEP is used as a high-energy phosphate donor to regenerate ATP, while ATP, GTP, CTP and UTP are directly supplied for transcription. In a ribose + NMP system, AMP, GMP, CMP and UMP are supplied instead, which can then be converted into NTPs by enzymes present in the cell extract. Ribose can also enter the cell extracts metabolic pathways and contribute to maintaining the energy and nucleotide pools. Therefore, the ribose + NMP system relies more on the metabolic activity of the cell extract, whereas the PEP + NTP system supplies more of the required components directly. The NMP-based system can also reduce the cost of the reaction because NMPs are cheaper than NTPs (Jewett et al., 2008)
3. Choose two cell-free reaction compositions from the literature, compare and contrast the choice of reagents and the concentration of reagents.
The two formulations are both based on E. coli cell extracts, but they use different strategies for energy regeneration and protein folding. Michel et al. (2012) was designed for disulfide-containing proteins, while Colant et al. (2021) focused on high protein production.
| Component | Michel et al. (2012) | Colant et al. (2021) |
|---|---|---|
| K⁺ source | 217 mM potassium acetate | 36 mM K-glutamate |
| Mg²⁺ source | 11 mM Mg(OAc)₂ | 4.8 mM Mg-glutamate |
| ATP | 1.2 mM | 0.48 mM |
| GTP/CTP/UTP | 0.86 mM each | 0.34 mM each |
| Energy source | 80 mM creatine phosphate + 5.8 μM creatine kinase | 13.2 mM PEP |
| Amino acids | 1.5 mM each | ~1.25 mM each |
| tRNA | 175 μg/mL | 68 μg/mL |
| Spermidine | — | 0.6 mM |
| Putrescine | — | 0.4 mM |
| CoA | — | 0.108 mM |
| NAD | — | 0.132 mM |
| GSH/GSSG | 2 / 5 to 10 mM | — |
| DsbC | 5 to 10 μM | — |
Overall, Michel et al. uses higher concentrations of the basic salts and NTPs and includes a creatine phosphate-based energy system and additional redox components (GSH, GSSG and DsbC) for disulfide-bond formation. Colant et al. uses lower NTP and salt concentrations but includes PEP, CoA, NAD and polyamines to support energy regeneration and metabolism. Thus, the reagent composition is adapted to the requirements of each CFPS system.
References:
- Michel, E., & Wüthrich, K. (2012). Cell-free expression of disulfide-containing eukaryotic proteins for structural biology. The FEBS Journal, 279(17), 3176 3184. https://doi.org/10.1111/j.1742-4658.2012.08697.x
- Colant, N., Melinek, B., Teneb, J., Goldrick, S., Rosenberg, W., Frank, S., & Bracewell, D. G. (2021). A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions. Biotechnology Progress, 37(1), e3062
Note: A recommended starting point would be to review a 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 (40 minutes)
1. Sign in to coopetition.htgaa.org and publish your first unique 20 µL CFPS reaction composition for each of the following (dont worry, you can resubmit later):
- sfGFP
- PETase
- Reteplase
For each composition, specify the reagents you would adjust from the relevant categories, including salts, energy sources, building blocks, cofactors, and redox components.
2. 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.
3) 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)
1. Build T7 compatible plasmids for cell-free expression using the amino acid coding sequences below:
sfGFP
PETase
Reteplase