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By John N.Abelson, Melvin I.Simon

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This chapter will focus on the lac operon fusion vector, MudJ, but any transposon that confers a selection will work. The method reported here relies on the creation of merodiploid strains in the form of tandem chromosomal duplications. Insertions into essential genes within the duplicated chromosomal segment are viable and are identified in a simple sectoring assay as colonies that are unable to generate haploid segregants in the presence of antibiotic selection. Tandem duplications arise spontaneously in bacterial and bacteriophage populations and can be present in a few percent of a growing population of cells (Anderson and Roth, 1977, 1981).

The site of insertion is identified by sequencing the junctions of the transposon and flanking chromosomal DNA. Striped box represents the transposon. Leftward and rightward arrows represent primers for sequencing transposon‐chromosome junctions. identification of essential genes in bacteria [4] 21 References Goryshin, I. , and Reznikoff, W. S. (1998). Tn5 in vitro transposition. J. Biol. Chem. 273, 7367–7374. Goryshin, I. , Hoffman, L. , and Reznikoff, W. S. (2000). Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes.

Plan of the Experiment Isolation of Mud Insertions in and Tn10‐araCþ Near Genes of Interest Mud: The random mutagenesis of Salmonella by MudJ transposition is described in Chapter 7. MudJ transposition is selected by selecting for MudJ‐encoded KmR. When the selection for MudJ insertions is done on rich medium, about 4% of the insertions are in auxotrophic genes. , 1980). For some pathways such as the his biosynthetic genes, all the genes are in a single nine‐gene operon; consequently, the chromosomal location of a his::MudJ insertion is known immediately.

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