Expression procedure

From CCP4 wiki

This is a suggestion given on the ccp4bb May 1st 2008:


What I usually do when I have a new construct is I run a cheap experiment with 2 x 50 ml TB cultures and measure the OD over time every hour. One culture is induced the other one not, at the end I compare the plots and deduce when it is best to induce and harvest my cells. The things I look at is what is the peak OD of the non-induced and induced culture, when does the non-induced culture reach halve height of their maximum (15 minutes before that timepoint is when I would induce them the next time, of course the aeration varies with volume , +/- antifoam, +/- baffeled flasks). It is not uncommon to see in the induced experiment that the maximum OD drops off at the end, if that is the case better harvest your cells at the peak as they start lysing. If your expression level is low that can have many factors and I won't go into that at this point. So if you don't have a growth curve make one, the longer you culture your cells the more your protein might be exposed to proteases that's another thing to consider when being lazy and growing over night cultures, know your system and act accordingly.

(I'll try to rephrase this in the near future and add some graphics. JB)