Raji
aside from the possibilites of toxic protein as already mentioned......
we had great results with overnight induction at 20oC for a protein that was
somewhat insoluble at 37oC. One thing that might work for you is to grow the
cells to OD 0.5 then lower the temperature to say 18 or 20. After an
hour or so
(retake OD) induce for overnight growth. Alternate is higher starting OD prior
to temperature reduction (i.e greater mass of cells) and shorter growth time
say 5-6 hours as you are thinking (we had protein that we only induced for 1
and a half hours too).
Hope that is useful!
Gina
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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.
