Week 3 HW: CFPS Coopetition

Copy and paste this section (section 3, A-F) into your website! Note, coopetition.htgaa.org is intended to support you as you answer the following questions.

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)

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

Magnesium glutamate is a cofactor, so ribosomes, RNAP, and all ATP-using enzyme need it. It is more sensitive to potassium glutamate, which usually sets ionic strength and keeps the ribosome and translation machinery stable. It has to be present in bulk.

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

All cell-free reactions need a steady supply of ATP and other nuceleotide triphosphates. For PEP+NTP, the nucleotides added are already charged. PEP gives its phosphate to ADP using the lysate enzyme pyruvate kinase. However, it’s expensive but gives a fast start. For the Ribose+NMP reaction, the nucleotides are uncharged and cheaper. The lysate enzymes burn the sugar to make ATP, and other lysate enzymes add phosphates onto the NMPs. This gives it a slower start but lasts longer. Since it relies on the lysate’s own metabolism, there are more variables to tune in the lysate. For cell-free reactions, Ribose+NMP reactions are cheaper based on cost-per-gram optimisation.

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

PANOx-SP and the coopetition baseline are built on the same core ingredients like lysate, HEPES buffer, glutamate salts, amino acids, an energy source, and nucleotides, but they have different purposes. PANOx-SP from Jewett & Swartz (2004) is a fixed recipe: it uses PEP as a direct energy source with NTPs, higher magnesium (about ~8 mM), and added cofactors and tRNA, all tuned for maximum protein yield.

The coopetition baseline is a lean starting point with fixed buffer, lysate, and DNA, leaving the remaining 11 µL open so you can choose cheaper energy sources (ribose, maltodextrin) and nucleotides (NMPs) and re-tune magnesium.

PANOx-SP optimizes for yield, while the coopetition optimizes for cost per gram.