I complemented a mutated gene in Staphylococcus aureus strain COL. The sequence is verified and everything seems to be right. The gene + flanking region is on a pCR2.1-TOPO + pSK265 shuttle vector. Total size is a little bit more than 8Kb.
The problem I have is that in phenotypic assays and sometimes in gene expression analysis, there's not enough complementation (only about 30-40% tops).
Any suggestions on why I am not getting good complementation? Any ideas of other plasmids that I can use as shuttle vectors will also be appreciated.