Sodium bicarbonate in extract
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Background: pH has been shown to be a contributing factor to the performance (output and longevity) of TX TL reactions and thus the additions of various buffers have been tested in the literature. Most buffering agents described in TX TL protocols in the literature are non native to the cell and thus may be having an unintended effects on enzymes or reactions that may be inhibiting protein production. Thus alternates were tested.
Experimental idea: The enzyme carbonic anhydrase is an enzyme that catalyzes the extremely fast interconversion of dissolved carbon dioxide and water and the ions bicarbonate and hydrogen. As pH drops, there is an increase in hydrogen ions in the media/cell/TX TL reaction that with the addition of bicarbonate, would allow for some neutralization of the pH as the hydrogen and bicarbonate would be converted into carbon dioxide and water.
Since carbonic anhydrase is ubiqutious, it should be present in most cell extracts prepared for TX TL and thus does not need to be added to the reaction.
N.B. Sodium bicarbonate is not easily dissolved into water and thus it is not possible to give a specific concentration for the stock solution. The user must define this themselves by adding a set amount of sodium bicarbonate into a given amount of water and then titring this solution into a TX TL reaction to determine a working concentration that works for them. The effects are the same.
Results: High amounts of sodium bicarbonate inhibited the TX TL reaction. Low amounts of sodium bicarbonate increased output substantially. Longevity was only slightly increased.
Also tested were potassium bicarbonate and also calcium bicarbonate. Neither produced similar or useful results.
