Construction of a plasmid for studies on the efficiency of Escherichia coli transformation by ligating the chloramphenicol acetyltransferase gene from pFM005 into the multiple cloning site of the plasmid pUC19

Abstract

To test whether the greater efficiency of transformation of pUC19 relative to pBR322 into Escherchia coli was due to differences in size rather than sequence, we normalized the sizes R of these two plasmids by inserting a 1.9kb cm gene into pUC19 to construct a 4.6kb plasmid (p3B) with the pUC19 replicon. By including a marker gene, different combinations of cell transformation events can be distinguished phenotypically. Phenotypic R and genotypic data suggest the ligation of the 1.9kb cm gene and pUC19 was successful. This was shown phenotypically by growth of a white transformed DH5 α colony on ampicillin and Xgal plates; and further confirmed genotypically using restriction enzyme digestion with Xba1 and gel electrophoresis. Although a ligation reaction was obtained, the expression of chloramphenicol resistance by the chloramphenicol acetytransferase gene could not be confirmed.