Hi Raji,
you can't compare cultures with only one protein expressed by amount of
cell yield or OD at the end. It's not really fun for the poor E.coli's
to produce our favourite proteins (even if they're not toxic to E.coli).
We keep thinking you just induce and harvest later E.coli takes care of
the rest. In reality expression is more a stressful situation it first
has to make sure to keep up with the various antibiotics you've added
then normal life ends for E.coli as soon as you induce as the
proteinmachinery is more or less exclusivly working for your protein.
What I usually do when I have a new construct is I run a cheap
experiment with 2 x 50 ml TB cultures and measure the OD over time every
hour. One culture is induced the other one not, at the end I compare the
plots and deduce when it is best to induce and harvest my cells. The
things I look at is what is the peak OD of the non-induced and induced
culture, when does the non-induced culture reach halve height of their
maximum (15 minutes before that timepoint is when I would induce them
the next time, of course the aeration varies with volume , +/- antifoam,
+/- baffeled flasks). It is not uncommon to see in the induced
experiment that the maximum OD drops off at the end, if that is the case
better harvest your cells at the peak as they start lysing.
If your expression level is low that can have many factors and I won't
go into that at this point.
So if you don't have a growth curve make one, the longer you culture
your cells the more your protein might be exposed to proteases that's
another thing to consider when being lazy and growing over night
cultures, know your system and act accordingly.
Juergen
Raji Edayathumangalam wrote:
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
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Sektion Naturwissenschaften
Universitaet Konstanz
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Tel. Lab : +49-(0)7531 88 3687
Fax: +49-(0)7531 88 2966
---------End of Included Message----------
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Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
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Seattle, WA 98195
Box 357742
Phone: +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch
