DNA gyrase improves DNA transformation of E.coli cells with large recombinant plasmids

摘要

To date, plasmids cannot accomodate large segments of foreign DNA without altering the efficiency of bacterial transformation. This becomes a limiting factor when the plasmids exceed 15 kb in size (1). We describe a method that increases the efficiency of transformation by using M.luteus DNA gyrase (topoisomerase II). which introduces negative supercoils into the recombinant plasmid (2,3). This technique was used to subclone different fragments of the mouse melanin-concentrating hormone (MCH) gene for which-standard protocols were poorly adapted (4). The size of the subcloned fragments varied from 4.1 up to 16.7 kb (Fig. 1 A). This method can be routinely used when subcloning of large DNA inserts within current plasmids are required.z