The development and application of a transposon insertion sequencing methodology in Escherichia coli BW25113

Robinson, Ashley (2017). The development and application of a transposon insertion sequencing methodology in Escherichia coli BW25113. University of Birmingham. Ph.D.

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Abstract

Escherichia coli is one of the most studied model organisms in biology. Even with decades of research, there are a substantial number of genes with an as yet unknown function. Previously, to determine the link between gene function and phenotype took significant experimental effort. However, newer methods are capable of providing large amounts of biological data in short timeframes. One such method, transposon insertion sequencing, is a powerful research tool, which couples transposon mutagenesis and next generation sequencing to identify genes that have important or essential functions. Here, three transposon insertion sequencing methods were compared. The techniques were adapted from previously published literature. Based on a number of metrics one technique was shown to be superior for data generation. This method was chosen for application in further transposon-insertion sequencing experiments. Subsequently, the optimised method was used to assess which genes were essential for the viability of the model organism E. coli K12. The results of this work were compared with the
literature and other databases of gene essentiality. A high degree of concordance was observed between our datasets and those generated previously through other methods. Indeed, the method described here was shown to have several benefits over previously used approaches.
Finally, genes involved with maintenance of the outer membrane were identified by using markers for membrane permeability in tandem with the chosen method. In keeping with previous literature multiple genes involved with many aspects of the cell envelope were reported. Many of the reported genes were shown to be involved with metabolic processes related to the biogenesis and maintenance of the cell envelope.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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