Dear Raji, I have expressed proteins in E. coli at 18ºC (and below that) with Amp+Kan+Chl and I've never had any problem with cell lysis. I usually grow cells at 37ºC and then cool the cultures down to the desired temperature before induction. I don't recomend growing your cultures at low temperature, specially having three different antibiotic resistences, as it may take a lot of time. Regarding expression time at 18ºC, I did had a protein that was expressed during only 6 hours at 17ºC and the yeld was reasonable. But normally I need longer expression times, sometimes even days. Hope it helps, Raquel
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 > >
