Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5→lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range.Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.The first sequence of bacterial chromosome was published more than 10 years ago. Now, hundreds of bacterial genomes are available for comparative genomics. The vast genetic information as well as introduction of new bacterial strains to industrial usage require the suitable tools for rapid genetic manipulations with a wide variety of bacterial species. Broad-host-range plasmids provide a valuable tool for such work. Numerous broad-host-range shuttle vectors, promoter-probe vectors, expression vectors and other special-purpose vectors are available now. The majority of them are based on the replicons of the IncQ or IncP groups [see for example [1-3]].Undoubtedly, RSF1010 can be considered as the most studied plasmid of this type. RSF1010 is a mobilizable, non