Jacob Before you spend you holiday weekend "dephaging" the lab and picking up on a comment from Artem and yourself, your protein could indeed be toxic to the cells. You do state in your email that the your are working with new constructs. What happens with your old constructs ? I've been unfortunate enought to work with a few toxic proteins in my time one of them being the notoroious bax protein (I hope your not working with this) that upon induction kills the bacterial cells (you get a flatline OD600nm). In fact if this protein is made in pLysS there is enough cell leakage that the lysozyme is released which then goes on to lyse other cells in the culture resulting in a lower OD then when you started induction and some very ugly looking pellets like you describe. If your protein is toxic to the cells you should be able to reach an OD600 of 0.6, likely more slowly than with a happy culture, and then when you induce with IPTG tracking the OD will tell you a lot. If it is seriously toxic, as in the case with bax, you can leave the cells to grow to a high OD without induction and work with the peanuts you'll get from the leaky expression off of the T7 promoter.
Stephen 2009/7/2 Jacob Keller <j-kell...@md.northwestern.edu> > Dear crystallographers, > > I recently expressed some new constructs, and found after my usual > expression protocol that the cell pellets were not compacted at the bottom > corner of the bottles us usual, but were instead smeared as a film on the > side, and further, were somewhat clumpy, like clots, and with a smaller > pellet in the usual location. The centrifugation was exactly as usual. I > noticed that there was also a bit more foam in the medium than usual, but I > am not convinced that this was the issue, although it might be a symptom. My > suspicion is that the constructs are lethal and cause cell lysis, but I am > not sure. Has anybody seen this phenomenon before, and gotten to the bottom > of it? > > Jacob Keller > > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > Dallos Laboratory > F. Searle 1-240 > 2240 Campus Drive > Evanston IL 60208 > lab: 847.491.2438 > cel: 773.608.9185 > email: j-kell...@northwestern.edu > ******************************************* >
