Abstract
The Keio single gene knockout collection comprises approximately 4000 mutants of E. coli K12 strain BW25113, where each mutant contains a kanamycin resistance cassette in place of a single nonessential gene. This mutant library has proven to be incredibly useful in the fields of bacteriology, chemical genomics, biotechnology, and systems biology, which is evidenced by the greater than 3800 citations that the article describing its construction has garnered in the approximate first 11 years since its publication. Among the various applications of the collection, the most extensive use has been in the assessment of how loss of specific gene function influences phenotypes. In this chapter, we describe pitfalls with use of the collection and procedures that can be employed to ensure robust phenotype assessment of mutations in the library. These include procedures for thorough confirmation of gene deletions by PCR, phage transduction of mutated loci to new host strains, and strategies for genetic complementation.
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This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI115075. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
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Aedo, S.J., Ma, H.R., Brynildsen, M.P. (2019). Checks and Balances with Use of the Keio Collection for Phenotype Testing. In: Santos, C., Ajikumar, P. (eds) Microbial Metabolic Engineering. Methods in Molecular Biology, vol 1927. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9142-6_9
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