Extract and energy polyA gels
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Background: Cell free reactions typically begin slowing down after 2-4 hours with increases of protein production very low by 4-8 hours. For building and testing larger circuits (such as multiple gene cascades) in cell free, it would be helpful to have reactions that last longer. A number of factors are thought to cause extract to die: depletion of fuel molecules (like ATP), accumulation of waste molecules (like ADP), change in pH, oxygene depletion (especially relevant for fluorescent reporters).
Experimental Idea: Can a polyacrylamide gel be made in the bottom of a plate with some portion of energy buffer instead of water in order to act as a reservoir for energy molecules and a siphon for waste molecules from a cell extract reaction?
Experimental Protocol: Polyacrylamide gels were made using varying amounts of APS, TEMED, Water, Energy Buffer and Acrylamide. These were left to sit (solidify) for 1-4 hours before a mixture of cell extract, energy buffer, and DNA containing a GFP reporter gene was added.
Results: Polyacrylamide gels seem to lower total expression by a factor of ~20 but also seem to increase longevity. It is unclear if the lowering of expression is due to the gel, toxicity of unpolymerized acrylamide which exists as a thin layer on the surface of the gel, or some other effect. APS and TEMED were found to actually increase cell extract fluorescence jointly (but decrease it individually).
Note: the TEMED APS experiments were run on different days using different BioTek plate readers, so the absolute output between the two graphs should not be compared.
