Thanks to everyone for all your suggestions. I am growing the cultures as we speak and have increased the temp to 22C and plan to harvest in about 6-8 hrs.
Thanks for the Q7 rule. I read it before but I couldn't remember exactly and a quick-and-dirty Google and Pubmed search did not bring it up. Let me clarify what I mean by lysis. Here are my observations: a) At the time of harvest, the final OD is lower for protein A + protein B (on two plasmids) than that for the same cells expressing only protein A or protein B (all else being similar at the time of induction). b) When the cells are spun down, the supernatant is cloudy and the pellet is smaller for A+B. The supernatant is clear for A alone or B alone. I am not sure this is a result of phage contamination since I have two other 'controls' for the same batch of competent cells in the same shaker, one containing just plasmid A and the other with only plasmid B. And, this is reproducible. Yes, I also very much suspect that my proteins may be a culprit, even though I only mentioned the antibiotics. Will see what happens this time. Thanks very much for all the helpful suggestions. Raji ---------Included Message---------- >Date: 30-apr-2008 12:30:50 -0400 >From: "Guenter Fritz" <[EMAIL PROTECTED]> >To: <[EMAIL PROTECTED]> >Cc: <CCP4BB@JISCMAIL.AC.UK> >Subject: Re: [ccp4bb] Bacterial induction at 18C > >Raji, > >> >> I am working with E. coli cells co-transformed with two plasmids and I find >> that my cells lyse >> following overnight inductions at 18C. >Sounds more like a phage contamination. The phage becomes active as soon >as the cells "energy level" decreases, e.g upon induction. We had once >the same trouble. If it is a phage, autoclave everything and clean the >lab thoroughly. >> I suspect (among many things) that Ampicillin+ >> Chloramphenicol+ Kanamycin in the medium may be the source of my woes. >> >> My colleagues have suggested growing cultures at 18C, say for 4-6h instead. >> Has anyone had >> reasonable protein expression levels by inducing cultures at 18C for 6h? >> From what I understand, the >> E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. >> >Rule of the thumb is the Q10 rule, or in the case of e.coli it is a Q7 >rule. Doubling decreases twofold when temperature eis decreased by 7 deg C. > >Godd luck, >Guenter >> I am already playing with lowering and/or doing away with the antibiotics. >> >> Any suggestions wrt 18C? The protein is insoluble at 30C. >> >> Thanks. >> Raji >> > >-- >*********************************** > >Priv.Doz.Dr. Guenter Fritz >Fachbereich Biologie >Sektion Naturwissenschaften >Universitaet Konstanz >http://www.biologie.uni-konstanz.de/fritz > >Universitaetsstrasse 10 >Postfach M665 >D-78457 Konstanz > >e-mail: [EMAIL PROTECTED] > >Tel. Office: +49-(0)7531 88 3205 >Tel. Lab : +49-(0)7531 88 3687 >Fax: +49-(0)7531 88 2966 > > > ---------End of Included Message----------
