Week 3 HW: Cell-Free Protocols

A. COMPARE CELL-FREE REACTION COMPOSITIONS

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 affect the cell-free reaction environment. Their optimal concentrations depend on the overall reaction formulation and the energy substrate being used. Jewett & Swartz (2004) reoptimized magnesium because PEP has a higher affinity for Mg2+ than pyruvate. PEP-based PANOx-SP used 20 mM Mg-Glu and 175 mM K-Glu, whereas pyruvate-based Cytomim used 8 mM Mg-Glu and 130 mM K-Glu

Magnesium glutamate is more sensitive to concentration changes. In Jewett & Swartz (2004) work, PANOx-SP changing Mg-Glu from 20 mM to 24 mM reduced relative protein expression from 100% to 73%, while K-Glu remained relatively stable around its optimum: 175 mM (100%), 200 (94%), and 225 mM (92%). In Cytomim, 8 mM gave the maximum yield, but 12 mM reduced it to 67% and 16 mM to 41%.

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

A PEP+NTP formulation supplies the cell-free system with nucleoside triphosphates (NTPs) such as ATP, GTP, CTP, and UTP. PEP serves as an energy phosphate donor to regenerate ATP, transferring its phosphate to ADP. For example, PANOx-SP (Jewett & Swartz, 2004) contains 33 mM PEP and 1.2 mM ATP plus 0.85 mM each of GTP, CTP, and UTP. This approach supplies transcription substrates, but PEP is relatively expensive, and its consumption generates inorganic phosphate.

In contrast, a Ribosome+ NMP formulation uses ribose together with nucleoside monophosphates (NMPs) such as AMP, CMP, GMP, and UMP. The lysate converts NMPs to NTPs. NMPs could support protein synthesis while reducing reagent costs.