I am attempting to knock out a gene in Rhizobium leguminosarum by crossover PCR and homologous recombination. I've cloned my insert into a sacB-containing suicide vector (pEX18), transformed into SM10 cells, conjugated into R. leguminosarum and identified merodiploids. I've grown the merodiploid colonies in broth where they should undergo the second cross-over and serially diluted and plated onto CR plates containing 5% sucrose. I'm screening these colonies by colony PCR and I am getting mostly WT bands and a few bands that have the mutant size and WT size band (i.e. merodiploids). Does anybody have any suggestions as to how the merodiploids are surviving on sucrose plates when the sacB gene should be killing them? How do I resolve this issue to get a mutant?
Thanks in advance!