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 (4.5-5.4kb) and carry various resistance determinants: kanamycin (Km(R)), tetracycline (Tc(R)) or chloramphenicol (Cm(R)). 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.