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
