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Re: [ccp4bb] Bacterial induction at 18C |
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CCP4bb navigationCCP4bb <-- 2008 <-- May 2008 <-- 01 May 2008Subject: Re: Bacterial induction at 18C From: Juergen Bosch jbosch {- at -} U {- dot -} WASHINGTON {- dot -} EDU Date: 2008-05-01 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" >>To: >>Cc: >>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: Guenter.Fritz@uni-konstanz.de >> >>Tel. Office: +49-(0)7531 88 3205 >>Tel. Lab : +49-(0)7531 88 3687 >>Fax: +49-(0)7531 88 2966 >> >> >> >> >> >---------End of Included Message---------- > > > -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch CCP4bb navigationCCP4bb <-- 2008 <-- May 2008 <-- 01 May 2008 |
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