A novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12.

We developed a novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12. The basic idea of the method is to randomly disrupt the genes on the DNA fragments cloned on the Kohara library by inserting a mini-transposon first, and then transfer the disrupted genes from the lambda vector to the E. coli chromosome by homologous recombination. Using this method, we constructed a set of 8402 Km(r) cis-diploid mutants harboring a mini-Tn10 insertion mutation and the corresponding wild-type gene on a chromosome, as well as a set of 6954 haploid mutants derived from the cis-diploid mutants. The major advantage of the strategy used is that the indispensable genes or sites for growth can be identified. Preliminary results suggest that 415 open reading frames are indispensable for growth in E. coli cells. A total of 6404 haploid mutants were deposited to Genetic Strains Research Center, National Institute of Genetics, Japan (Chapter 26) and are available for public distribution upon request (http://shigen.lab.nig.ac.jp/ecoli/strain/nbrp/resource.jsp).