E coli die after inducer - (Jul/21/2005 )

Hi everyone!
I am trying to do a protein expression experiment but my E coli start to die as soon as I give them the inducer (anhydrotetracycline). I have used this system before with really nice overexpression. I believe that both the concentration of the inducer and the bacteria are ok! Any ideas about what might be happening?
Thanks for helping out!
ACunha

You said you used the system before - are you saying that you're trying to express the same construct that was successfully expressed previously?

If it is the same construct, have you checked for mutations? You're using the same bacterial strain? Same growth temperature and media (components and pH)?

I am using a different construct but everything else is the same, including the vector used for the construct!

It is very possible that the new construct is lethal to the cell. It happens quite frequently with foreign proteins. The best advice I can give is to get the BL21(DE3) pLysS strain. We've had some luck expressing toxic proteins in this strain.

http://www.stratagene.com/products/display...ct.aspx?pid=553

If you're expressing for purification, it usually doesn't matter if most of your cells die, so long as they produce sufficient quantity for your purposes before they kick it. I'd do a test expression varying temperature and length of induction to optimize your expression output.