Analysis of Potential Essential Genes Using the Escherichia Coli K-12 Keio Mutant Collection

Darryl Dague

Date of Award

2009

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Keywords

E. Coli, Essential Genes, Keio Collection, Transduction, Escherichia Coli

Area of Concentration

Chemistry

Abstract

The identification of essential Escherichia coli genes furthers our understanding of fundamental cellular processes. It is of practical importance because it reveals a set of targets that facilitate the discovery of antibiotic compounds. Unfortunately, the current literature leaves us with conflicting results as to the essentiality of many E. coli gene products. Dr. K. Rudd's laboratory set out to resolve these discrepancies and identify the complete essential gene set. The present paper reports the results of my contribution to this project. As a starting point, we examined the Keio collection, a resource of 3985 single-gene knockouts. We used P1 bacteriophage transduction to transfer these knockout mutations from the mutagenized background to our wild-type strain. The transduction frequency is inversely proportional to the missing gene's essentiality. For the majority of Keio mutants, we verified the non-essentiality of the disrupted gene. In cases where transductions failed, the mutants were investigated further. We circumvented factors that mask the 'true' phenotype of an essential gene deletion (e.g. tandem duplications, polar effects, and suppressor mutations). In addition, we redesigned a number of mutations whose construction was faulty in the Keio collection. The project was successful insofar as it verifies that the phenotype of each Keio strain is the result of its respective gene deletion, irrespective of the chromosomal background. As will be shown, P1 transduction is particularly applicable to this end.

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