A modified unique site elimination mutagenesis in constructing a chloramphenicol resistance-encoding pGEM vector.

Recombinant DNA research requires new and versatile cloning vectors. Many of the popular plasmid vectors, such as the pUC (4), pTZ (Amersham, Arlington Heights, IL, USA) and pGEM (Promega, Madison, WI, USA) series, share common characteristics, including high copy number, sizes of around 3 kbp and the presence of a multiple cloning sequence (MCS) within the coding sequence for a part of the lacZ gene isolated from E. coli. Cloning a fragment using a restriction site within the MCS of one of these plasmids allows blue/white selection for recombinant molecules. These plasmids also carry a gene for ampicillin-resistance. In our hands, selection for ampicillin-resistant bacteria can be problematic, as many nonresistant, satellite colonies are often found surrounding the actual resistant colonies. Although this problem can be overcome using shorter incubation periods, the use of chloramphenicol selection does not present such problems. Since the majority of these plasmids are around 3 kbp in size, this can lead to problems when trying to clone fragments, which are also close to 3 kbp. Subsequently, it can also be difficult to separate the desired cloned fragment from the vector fragment. Site-directed mutagenesis has become a major force in the analysis of gene function. The ability to precisely change a single base pair within a segment of DNA has proven invaluable— illuminating the effects of single amino acid changes in a protein. One common method for site-directed mutagenesis is the unique site elimination (U.S.E.) protocol (1). We have introduced the gene encoding chloramphenicol resistance into the plasmid pGEM-7Zf(+) (Promega), and in the process, disrupted the gene for ampicillin resistance. This has increased the size of the plasmid to 4 kbp. However, the inserted fragment introduces a number of restriction sites rendering some sites in the MCS unsuitable for cloning. We describe the construction of a chloramphenicol resistance encoding pGEM7Zf(+) derivative and a modification of the U.S.E. protocol that does not require elimination of a unique restriction site. The gene encoding chloramphenicol resistance, chloramphenicol acetyl transferase (CAT), was isolated as a blunt-end SmaI fragment from pCAT19 (2) using a low-melting point agarose gel and β-agarase treatment. The vector pGEM-7Zf(+) was cut with ScaI within the bla gene encoding β-lactamase to give blunt ends. The SmaI CAT fragment was ligated to the ScaI vector ends, and the ligation mixture transformed into E. coli strain JM101 with selection for chloramphenicol resistance. Recombinant plasmids with the CAT gene inserted in both of the two possible orientations were isolated. One of these, pKM1 was chosen for the