hi to all,
i have noticed that if i incubate two parallel bacterial cultures in the same conditions ( one where i add IPTG for protein induction and another in which I don't add anything) the OD of the the uninduced culture is higher than the one of induced cultures.
so i'm wandering if a decrease of OD could be a clue for protein expression.
Yes, you are right. When protein biosynthesis mechanisms are working at maximum level (like under induction with IPTG) the proliferation rate is slowing down. It is a general rule for proteins in bacterial cultures secreted under induction (not a constitutive way of secretion).
It may be a clue if significant cellular resources are directed towards heterologous protein expression. Sometimes we notice this (association between relatively lower culture OD and heterologous protein expression) and other times (when expressing potentially toxic proteins using low IPTG concentration) not.
ok, perfect. the problem is that when i prepare the samples for the SDS-PAGE, i can't see a difference between the induced and the not induced, although the difference in the growth tate between the two cultures
How big (and reproducible) is the OD difference between induced and uninduced cultures?
Perhaps you subject the bacterial lysates to Western blot.
Depends on the strain. In DE3 lysogens, IPTG leads to T7 RNA polymerase expression, which hijacks transcriptional control in the bacteria to T7-dependent (not host) promoters. Thus, in DE3 strains, IPTG greatly slows cell growth by suppressing expression from the E. coli genome, and will have this effect irrespective of whether your transgene gets properly expressed.
Hi...
I agree to what Daniel has said. As a matter of fact, you can also see a difference in pellet color of induced and uninduced cultures (pellets of induced cultures are much lighter in color). I also think the difference in OD could be an indicator of protein expression, however it is completely arbitrary. IPTG itself puts cells under metabolic stress so obviously the cell growth slows down when IPTG is added.
However, if you know for sure the gene of interest is always getting expressed using same host-vector combination, then you can consider the difference of OD as an indicator of protein expression. But it can't always be trusted, as Daniel said, particularly when the expression conditions are changed.
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