Abstract
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 λ vector to the E. coli chromosome by homologous recombination. Using this method, we constructed a set of 8402 Kmr 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).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kohara, Y., Akiyama, K., and Isono, K. (1987) The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50, 495–508.
Jishage, M., and Ishihama, A. (1997) Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110. J. Bacteriol. 179, 959–963.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Frschauf, A.-M., Lehrach, H., Polstka, A., and Murray, N. M. (1983) Lambda replacement vectors carrying polylinker sequences. J. Mol. Biol. 170, 827–842.
Sanger, F., Niklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463–5467.
Kleckner, N., Bender, J., and Gottesman, S. (1991) Uses of transposons with emphasis on Tn10. Methods Enzymol. 204, 139–180.
Ried, J. L., and Collmer, A. (1987) An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis. Gene 57, 239–246.
Ohshima, T., Aiba, H., Baba, T., Fujita, K., Hayashi, K., Honjo, A., et al. (1996) A 570-kb DNA sequence of the Esherichia coli K-12 genome corresponding to the 28.0–40.1 min region on the linkage map. DNA Res. 3, 137–155.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press Inc., a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Miki, T., Yamamoto, Y., Matsuda, H. (2008). A Novel, Simple, High-Throughput Method for Isolation of Genome-Wide Transposon Insertion Mutants of Escherichia coli K-12. In: Osterman, A.L., Gerdes, S.Y. (eds) Microbial Gene Essentiality: Protocols and Bioinformatics. Methods in Molecular Biology™, vol 416. Humana Press. https://doi.org/10.1007/978-1-59745-321-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-59745-321-9_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-378-7
Online ISBN: 978-1-59745-321-9
eBook Packages: Springer Protocols