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Background  RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet.    Results  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 P  lac UV5 → 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 P  lac UV5  by any desirable promoter for construction of novel derivatives with changed copy number or host range.    Conclusion  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.

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