Week 3 HW: Cell-Free Protocols

Assignment A:

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

Potassium glutamate and magnesium glutamate serve different purposes in a cell-free reaction. Potassium glutamate primarily helps reproduce the ionic environment of the E. coli cytoplasm, where potassium is the predominant intracellular cation. Magnesium glutamate supplies Mg²⁺, which is essential for processes involving ATP, nucleotides, ribosomes, and other components of transcription and translation. The concentrations therefore need to be balanced rather than simply maximized: changing them alters the chemical environment in which the cell-free machinery operates.

The concentrations differ between CFPS formulations because different lysates and reaction chemistries have different optima. For example, PANOx-SP formulations commonly use potassium glutamate at roughly 130–175 mM and magnesium glutamate around 10–20 mM, illustrating that the two salts operate at very different concentration ranges. More recent formulation work likewise treats potassium glutamate and magnesium glutamate as independently tunable variables rather than assuming a fixed ratio.

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

A PEP + NTP formulation supplies pre-made nucleotide triphosphates (ATP, GTP, CTP, and UTP) for transcription and uses phosphoenolpyruvate (PEP) as a high-energy phosphate donor to regenerate ATP. In other words, the reaction starts with the expensive, ready-to-use forms of the nucleotides and an energy source that can rapidly regenerate ATP. The course materials identify PEP as an energy-regeneration substrate and NTPs as the building blocks for RNA synthesis. Recent work describes PANOx-SP as a phosphorylated-energy-substrate formulation in which PEP is consumed to generate ATP, producing a rapid initial ATP surge.

A Ribose + NMP formulation takes a more metabolic approach. Instead of supplying all of the nucleotide triphosphates directly, it supplies nucleoside monophosphates (AMP, CMP, GMP, UMP) and relies on enzymes remaining in the E. coli lysate to convert these lower-energy precursors into the NTPs required for transcription. Ribose provides another metabolic precursor that can feed nucleotide-salvage and related pathways. This approach therefore asks the lysate to do more of the work of regenerating the reaction’s nucleotide and energy pools. The 2026 Olsen et al. study found that formulations based on NMPs and inexpensive metabolic substrates could sustain protein production while substantially reducing reagent cost relative to PANOx-SP.

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

I chose PANOx-SP from Jewett & Swartz (2004) and the optimized reagent formulation (RFopt) from Olsen et al. (2026) because they represent two very different approaches to cell-free protein synthesis.

PANOx-SP is a relatively complex, high-yield formulation based on a phosphorylated energy substrate. It uses PEP at 33 mM to regenerate ATP, along with pre-supplied nucleotide triphosphates (ATP at 1.2 mM and GTP, UTP, and CTP at 0.85 mM each). It also contains potassium glutamate, magnesium glutamate, ammonium glutamate, amino acids, folinic acid, tRNA, NAD, CoA, spermidine, putrescine, and oxalate. The original formulation reported by Jewett and Swartz used 175 mM potassium glutamate and 20 mM magnesium glutamate.

In contrast, RFopt was developed specifically to reduce the cost and complexity of CFPS. Olsen et al. screened 1,231 formulations and arrived at a 12-component optimized formulation that uses non-phosphorylated metabolic substrates rather than PEP. A major change is the replacement of expensive NTPs with nucleoside monophosphates (NMPs), together with potassium glutamate and amino acids. The optimized formulation also incorporates components such as ribose, phosphate, and HEPES rather than relying on the large collection of cofactors and secondary energy substrates used in PANOx-SP.

Assignment B:

Part 1.

My cfps composition table.

K-glutamate — 340 mM

I’m keeping it high because the optimized low-cost formulations found a high-K-glutamate regime useful for protein production.

Mg-glutamate — 8.22 mM

Magnesium is needed by the molecular machinery, but it is more sensitive than potassium. Therefor I decided not to increase it yet.

Amino acids — 3.25 mM

The building blocks of the protien. Needed.

AMP/CMP/GMP/UMP

The lysate has metabolic machinery that can turn them into the triphosphate forms needed for RNA synthesis. That’s one of the main tricks behind the cheap formulation.

I designed my first sfGFP reaction around the low-cost, non-phosphorylated strategy reported by Olsen et al. rather than the PEP-based PANOx-SP formulation. I used potassium glutamate, magnesium glutamate, amino acids, and NMPs while omitting the PEP, NTP, NAD, CoA, and other ancillary components of the conventional formulation. My goal is to reduce reagent cost while maintaining sufficient protein production. I used an RFopt-like composition as my starting point and made a small variation in potassium glutamate concentration to create a distinct candidate for screening. Olsen et al. reported 2.4 ± 0.3 g/L sfGFP at approximately $60/g protein using their optimized formulation.

Part 2.

I upvoted FkpA because it could complement DsbC in the Reteplase reaction by addressing a different part of the protein-folding problem. FkpA is an E. coli chaperone with peptidyl-prolyl isomerase activity. Groff et al. found that adding FkpA to cell-free antibody synthesis reduced aggregation and improved protein solubility and folding, while DsbC primarily improved disulfide-bond formation and assembly. The two therefore addressed different folding bottlenecks and showed complementary effects.

Reteplase is particularly attractive as a target for this idea because it contains nine disulfide bonds and requires an oxidizing CFPS environment with DsbC for correct folding. Although FkpA has not been demonstrated in the cited study specifically for Reteplase, I think it is a worthwhile reagent to test because it could reduce aggregation and improve folding in addition to the disulfide-bond assistance already provided by DsbC. I would therefore treat its benefit for Reteplase as a hypothesis to test, rather than assuming that the antibody results will transfer directly.