Construction of pANPT, a Novel pBR322-derived Plasmid with Deletion of the Tetracycline Resistance Gene

Previous studies have shown that despite belonging to the same family of ColE1-type plasmids, the pUC19 plasmid is selectively maintained after a few generations when co-transformed with the pBR322 plasmids into Escherichia coli DH5α host cells. Some intrinsic differences between the pUC19 and pBR322 plasmids were proposed as potential explanations, including the size difference of 1675 base pairs between the two plasmids. In this study, we initiated an investigation of the role of plasmid size on the exclusion effect by creating a pBR322 plasmid mutant through the deletion of the tetracycline resistance gene. Inverse PCR with primers designed to specifically direct outwards from the tetracycline resistance gene was employed, creating a product that was ligated to generate this 3173 bp deletion mutant. The identity of the construct created was then confirmed through diagnostic and physiological tests. Even though the calculated size did not precisely match the expected size, relative size comparison as well as the inhibition of growth on tetracycline supplemented media supported the construct identity as a tetracycline gene deletion mutant, named pANPT. This pBR322-derived novel plasmid with size comparable to that of the pUC19 plasmid can be used to explore the role of size on the exclusion effect during the cotransformation of the pUC19 and pBR322 plasmids.