Hi Hannes, We have recently had a batch of phage contamination - one of the symptoms was exactly as you described, when pelleting the cultures, a smeary, fluffy pellet appeared. The other symptoms we had were that during growth, the OD would rise up until ~0.5, then drop, a foam would develop on top of the media (that remained upon standing) and if you examine the flask by eye there are ~2 mm strands floating around in the (almost clear) media.
We found that this occurred (apparently) randomly within batches of the same media (using the same starter culture, same aliquot of antibiotics, IPTG etc), which ruled out a problem with the central media kitchen. After asking around, we found that other people in our building had experienced similar problems. The general consensus was that there was probably some airborne phage floating around in the incubator, which was getting in when flasks were open. At that point we made sure that we never opened our flasks in the incubator, so every time we do an OD or need to open the flask, we remove it, bring it back to our lab (a different room) and open it under a flame. Since then we have not had a single flask die. Although it is a bit of a pain to have to remove flasks every time you take an OD or induce, it is less of a pain than watching your media slowly die (especially if you like doing NMR, and have expensive multiple-labelled media...) Best Regards, Tom On Wed, May 12, 2010 at 9:29 AM, Hannes Uchtenhagen <hannes.uchtenha...@ki.se> 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 > -- Skype: tom.murray.rust +44 7970 480 601 (UK Number) +61 41535 4486 (Aussie Mobile)
