Extracts and dialysis membranes
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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: by placing a reaction chamber (10uL) in contact with a reserve of energy buffer separated by a semi-permeable membrane that allows for small molecules (ATP, ADP, H+) to cross, but not proteins, it might be possible to use passive transport to continuously supply new fuels and continuously sequester waste molecules.
Results: This did not work, likely because the membranes tested are somehow toxic to extract.
Similar tests were tried on two different kinds of membranes: Dialysis Tubing, 10K MWCO, 16 mm and the membranes taken from MINI Dialysis Device, 10K MWCO, 0.1 mL. Presoaking the membranes in DI water for an hour did not substantially change the results. Adding Junk DNA into the mix (under the assumption that maybe DNA was sticking to the membranes) also did not change the results.
