Hi Raji, Any possibility that your protein lyzed the E. coli. Some proteins do that. It's unlikely the antibiotics are the problem. You have to find out when the bugs start to lyze and harvest before that time point. In addition, lower the induction temperature further down in case the lysis is due to the enzymatic activity of your protein. We actually had a case that making an inactive mutant solved the problem.
Good luck! Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 [EMAIL PROTECTED] www.accelagen.com -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Raji Edayathumangalam Sent: Wednesday, April 30, 2008 8:40 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Bacterial induction at 18C Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. 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. 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
