Cloning the Potential Insertion Site of a Promoterless Kanamycin Resistance Cassette Recombined by the Lambda-Red Recombinase System

Abstract

A previous study by Jaeger et al. attempted to disrupt the lac I gene by using the R lambda red recombinase system to insert a promoterless kanamycin resistance (Kan ) cassette into the gene. Instead of the expected deletion of the lac I gene, subsequent study found that the insert actually occurred outside lac I. Our study started to map the R insertion site of the Kan cassette by constructing a genomic library of the mutated E.coli R C29 cells using the pUC19 vector. To screen for relevant clones which contain the Kan R cassette, a double digest using two restriction enzymes which cut within the Kan gene was performed. Clones that showed the expected 400 bp fragment were screened for kanamycin resistance. Our study have identified 5 clones which potentially have pUC19 R carrying a Kan cassette flanked by the insertion site.