Novel broad-host-range vehicles for cloning and shuffling of gene cassettes

Novel vectors for cloning and shuffling of gene cassettes based on minireplicon of broad-host-range RA3 plasmid from IncU incompatibility group were constructed. A series of minireplicon variants were prepared with copy number ranging from low (1–2 copies per chromosome), medium (10–15 copies per chromosome) to high copy number (80–90 copies per chromosome). The new cloning vectors are relatively small in size (4.5–5.4 kb) and carry various resistance determinants: kanamycin (KmR), tetracycline (TcR) or chloramphenicol (CmR). The vectors were engineered to facilitate cloning and shuffling of the functional modules with or without transcriptional terminators. Using the described strategy, a bank of functional modules, ready for exchange, has been initiated.

► Novel broad host range cloning vectors based on RA3 of IncU group were constructed.
► Different copy number variants with various resistance determinants were generated.
► The vectors facilitate adding transcriptional terminators to the functional modules.
► Vectors enable gene cassettes shuffling to build synthetic plasmid of new properties.