This story is rather puzzling indeed, and it is difficult to see why in case of toxicity one would have transformation problems with that one strain in particular.
Also, I am wondering how likely it is that a combination of toxicity and leaky expression would lead to transformation problems in the first place. I have quite a bit of experience with the expression of extremely toxic proteins/peptides and I usually find that most of the popular E. coli strains are quite capable of somehow getting rid of the gene or its expression if they really need to, while retaining full antibiotic resistance (the upshot of which is a normal transformation efficiency but zero expression upon induction). My advice would be to first check very carefully for (extremely) trivial reasons for the failed transformation. Best wishes, Sebastiaan Werten. ----- Original Message ----- From: Cynthia Kinsland To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, May 04, 2009 4:52 PM Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone Using pQE30, any E. coli is an expression host. Because it uses the T5 promoter, you don't need an E. coli strain carrying the T7 RNA polymerase (so, you don't need a "DE3" strain). As noted by Artem, you are most likely having a leaky expression problem. However, it is odd that DH5a will transform if this is the case since it does not carry the lacIq mutation. XL1-Blue does, which is why it was suggested below. You could try expression right in DH5a, since you have the plasmid there. Your transformation difficulties seem strange. Using this vector, DH5a should not transform any more stably than the other strains you tried.
