Let me start by saying I'm a graduate student and I'm trying to create an isogenic mutant. I'm using a suicide vector that should experience a campbell-like recombination event to delete my gene of interest. The first event inserts an antibiotic resistance marker, which I see. However, when I induce the second event, I get bacteria growth, but everything comes up wild-type. Everything I come up with to explain this, doesn't make any sense. Any ideas?
THe one I'm hoping isn't the case is that the mutation is lethal. In that event, other than a conditional mutant (which I'm researching information on since this hasn't been attempted as far as I can tell with my bacteria), what else can I do to study gene function other than put it in E. coli and study it?
THanks in advance for anything that points me in the right direction.