Homework 3 – Designing a Cell-Free Reaction Composition

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 potassium glutamate and magnesium glutamate differ between formulations because CFPS performance depends on the chemical environment, so reagent concentrations need to be optimized together. Magnesium appears to be more sensitive because its concentration may need to be re-optimized for different lysate batches, and the reading also notes that protein synthesis depends nonlinearly on free Mg²⁺ and nucleotide concentrations.

  2. Explain how a PEP+NTP reaction formulation is different from a Ribose+NMP reaction formulation. A PEP + NTP reaction uses PEP as an energy source and NTPs as the building blocks for producing RNA. NTPs have three phosphate groups, which makes them a more ready-to-use form, but they also come with a higher reagent cost. In comparison, NMPs serve a similar role but have only one phosphate group, so they are not as ready-to-use as NTPs. Ribose is a sugar that can be used as a more basic starting material. Since the lysate still contains metabolic machinery from the original cells, it can help convert these simpler starting materials into the forms needed for protein synthesis.

  3. Choose two cell-free reaction compositions from the literature, compare and contrast the choice ofreagents and the concentration of reagents. I compared PANOx-SP and RFopt. For the reagents that they both use, RFopt has 8 mM magnesium glutamate, 362 mM potassium glutamate, and 5 mM amino acids, while PANOx-SP has 20 mM magnesium glutamate, 175 mM potassium glutamate, and 2 mM amino acids. They also use different reagents for energy and nucleotides. PANOx-SP uses 33 mM PEP and NTPs, while RFopt uses 50 mM ribose and NMPs. overall, i would claim PANOx-SP relies more on directly supplied, ready-to-use energy and nucleotide components, while RFopt uses more basic starting materials and relies more on the metabolic activity remaining in the lysate.

B. DESIGN ONE REACTION COMPOSITION FOR EACH OF THE THREE PROTEINS (40 minutes)

  1. I reduced PEP(from 30 to 20) from the reference condition and supplemented the reaction with glucose(5.55) to test whether sfGFP production can be maintained while lowering reliance on the more expensive PEP energy system. I wasn’t assuming a stoichiometric replacement between PEP and glucose. I reduced PEP by one-third and added a moderate amount of glucose as a first screening condition to see whether glucose can partially support energy regeneration while reducing PEP use.
  2. I kept the PETase reference formulation and added GSSG and GSH because PETase needs to fold into the correct shape to work properly. These reagents may help create a better chemical environment for that folding process, while DsbC helps the protein form the right internal bonds. I want to test whether this improves PETase activity. I chose GSSG and GSH(they work as a redox pair) because PETase needs to fold correctly to be active, and this glutathione pair is commonly used in cell-free systems to create a chemical environment that supports disulfide-bond formation. I kept DsbC as the folding helper and used GSSG/GSH to adjust the surrounding folding environment.
  3. I kept the established Reteplase folding system unchanged and instead modified the energy source. I reduced PEP from the reference condition and added sodium pyruvate to test whether part of the more expensive PEP system could be replaced while maintaining Reteplase activity.
  4. I upvoted FkpA because Reteplase is the most difficult of the three target proteins to fold, with nine disulfide bonds. The proposed rationale suggests that FkpA could provide additional folding support together with DsbC. I thought this was especially relevant because the current Reteplase formulation already includes DsbC.