Week 3 HW: Cell-Free Protocols

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

K-Glu supports the general conductive/ionic environment of the cell-free lysate, which has a direct effect on ribosome, enzyme, and protein–nucleic acid binding (ex. transcription) function. The concentration range for effectiveness is broad. Mg-Glu supplies magnesium ions that directly bind to ribosomes, tRNAs, TX/TL enzymes, and ATP/NTPs. Mg-Glu concentration is highly sensitive, and this concentration is far more sensitive.

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

A PEP+NTP is the definition of paying a lot of money upfront for highly energetically charged intermediates that can supercharge a CFPS reaction to get high titer/yields. However, the cost is prohibitively expensive to support sustainable, realistic biomanufacturing production.

Meanwhile, a Ribose+NMP fed reaction is an example of a CFPS reaction where you feed relatively inexpensive reagents and rely on a well-balanced, well-stabilized, and energy-regenerating metabolic network to still power a CFPS reaction. Many reaction conditions come into play here, such as salt and pH buffering reagents, dissolved oxygen supply, and other cellular compositions.

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

I’ll choose to compare the first major reaction formulation, PANOx-SP with the AI-optimized formulation from Ginkgo/Open-AI. For one, PANOx-SP used expensive PEP as the energy-charged intermediate/phosphate donor and directly supplied expensive NTPs. The AI formulation provides less-expensive ribose and NMPs and relies on the CF metabolism to recharge these molecules.

Another difference is that the Ginkgo/Open-AI formulation has very high K-Glu concentrations and allows for some HEPES-KOH pH buffer solution, supporting the idea that salt requirements and pH conditions greatly affect protein production and energy regeneration. PANOx-SP had zero HEPES and much lower K-Glu conditions.

A final difference is that the AI formulation was able to supply zero ATP or NTs, nor any NAD/CoA, all of which are very expensive and were used in PANOx-SP. This is a big breakthrough for allowing the cell’s metabolism to build up a robust protein production environment.