Dear Hannes, I am afraid it can still be phage problems. There are many kinds of phages that infect E. coli and some of them behave like you describe: not always lysed cells but from time to time and coming and going. The phage may anyway be present but in its lysogenic state and only entering the lytic pathway under stressful conditions.
Try some strains that lack or have mutated form of a common phage receptor: fhuA. I know that for example NEB sells a BL21(DE3) alternative (T7 Express) with a mutated fhuA (fhuA2). This strain (T7 express) also has the benefit of lacking the lambda prophage so that if your expressed proteins (esp. the inclusion bodies) trigger the SOS response you will not have lambda phage-caused lysis at least. Best regards, Martin PS. I am not affiliated to NEB in any way.... On May 12, 2010, at 10:29 AM, Hannes Uchtenhagen wrote: > Dear ccp4bb, > > We used to routinely overexpress a number of mammalian proteins to inclusion > bodies in E.coli. Lately however we get from time to time smeared and small > or no pellets after spinning down the cultures (lysed I assume). > I have read (with some horror) previous threads pointing at phage > contamination and toxic proteins. I am doubtful about the phages as the > problem is not expanding and very frustratingly it appears seemingly random, > coming and going. Finally the one time I checked I did not see any plaque > formation on a e.coli plate tested with a lysed culture. > We have expressed the proteins before many times to inclusion bodies without > any problems. I still could imagine that they could be toxic when soluble. > But why would suddenly happen since we did not change any conditions that we > are aware of? > > It would be really great to get some of your ideas on what else could cause > this kind of problem (IPTG, aeration, leftovers from glassware cleaning, > media) or if I missed something with respect to the phages. > > Many thanks for your time! > hannes uchtenhagen > > > -- > Hannes Uchtenhagen > Karolinska Institutet > Center for Infectious Medicine (CIM) > Karolinska University Hospital Huddinge, F59 > SE-141 86 Stockholm, Sweden > > Office: +46-(0)8-524 86981 > Mobile: +46-(0)7-36901461
