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" <[log in to unmask]>
>To: <[log in to unmask]>
>Cc: <[log in to unmask]>
>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
>Fachbereich Biologie
>Sektion Naturwissenschaften
>Universitaet Konstanz
>http://www.biologie.uni-konstanz.de/fritz
>
>Universitaetsstrasse 10
>Postfach M665
>D-78457 Konstanz
>
>e-mail: [log in to unmask]
>
>Tel. Office: +49-(0)7531 88 3205
>Tel. Lab : +49-(0)7531 88 3687
>Fax: +49-(0)7531 88 2966
>
>
>
---------End of Included Message----------
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