Week 3 HW: Cell-Free Reaction Design

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?

The compositions require differnt pH conditions. Varying the concentrations of potassium glutamate and magnesium glutamate helps modify the pH across compositions. It’s also stoichiometrically helpful to have a compound that delivers two glutamates vs. one.

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

A PEP+NTP reaction uses PEP to supply phosphate groups and replenish those of spent NTPs. A Ribose+NMP reaction instead uses ribose to build new nucleotides and uses pure phosphate as its phosphate source.

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

The biggest differences between the 2004 and 2026 formulations (see below) are that the 2004 version supplies extra phosphate groups while 2026 relies on the existing compounds and that the 2004 uses only NTPs while 2026 uses a combination of NMPs (which can be converted to NTPs) and NTPs.

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)

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

Note: if you’re unable to login with your HTGAA keycloak account, please post in Zulip so that you can participate in the coopetition!

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

See submission on coopetition site.

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.

TrxA can act as an oxidizing protein that facilitates disulfide bond formation and could improve synthesis efficiency for reteplase. See paper here: https://pmc.ncbi.nlm.nih.gov/articles/PMC11180911/

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.