Why can I get Mesorhizobium loti or Rhizobium sp. NGR234 without pCAMBIA1105.1R in it?
In both cases the strain was first transformed with pCAMBIA1105.1R by electroporation and the modified Ti plasmids pWBTi1 or pWBTi3 were introduced next by triparental mating from Sinorhizobium meliloti. You should be able to cure these bacteria of the binary plasmid by growth in non-selective conditions (no spectinomycin or streptomycin) for some generations, followed by replica plating on media with and without spec (or strep) to screen for colonies that have become sensitive to it. A quicker method would be to transform in another pCAMBIA plasmid (or other plasmid with the same backbone such as the pPZP plasmids) and select for its resistance gene. This should “bounce out” pCAMBIA1105.1R since cells don’t like to keep two different plasmids with the same replicon. You should still screen for loss of spec/strep resistance though, to make sure. We don’t intend to do this ourselves. We are busy working on a broad host range Ti plasmid and this will be made available in strains without
