Leaky Expression? - (Jun/09/2008 )
Hi, im posting again, i like this forum.
Anyways has anyone had a problem wiht leaky expresssion. I mean when i induce my BL21 star cells with IPTG and i monitor expression i see that my uninduced cells also have a band where the expression should be even though the band is getting larger with time induced. Just wondering if the cells expresses a little protein without IPTG. Thanks
BL21 cells will have a baseline expression of your protein without the presence of IPTG. You should however see a huge difference in the induced samples. As a control though, you should make sure the BL21 cells do not express a native protein (without being transformed) at the same size as your protein, it could be a native protein. But if it isn't native and your cells are expressing a baseline amount, I wouldn't worry about it, as long as you get induction.
It's a known problem that's why some people use strains containing a repressor plasmid like Tuner pLAC which is supposed to block the basal expression specially if the protein might be toxic to E.Coli cells
You can grow the cells with a low level of glucose or even glycerol, then when you're ready to induce either add IPTG and start, or if you want to be really cautious, spin down the cells and resuspend in media without glucose/glycerol.
You can also try full-on autoinduction. There are a number of autoinduction media you can buy prepared, but the recipes are fairly straightforward. Check [attachment=4815:studier_et_al_2005.pdf] by Bill Studier.
This baseline expression isnt a problem is it? I was wondering there was a band in uninduced cells when there shouldnt be thats all.
Baseline expression of endogenous proteins won't be a problem 99% of the time. If it's your protein of interest, the only issue might be toxicity, but if it's not toxic to the cell, then you're right, there's no real problem.