Using TraDIS to probe the model organism Escherichia coli

Goodall, Emily C. A. ORCID: https://orcid.org/0000-0003-4846-6566 (2019). Using TraDIS to probe the model organism Escherichia coli. University of Birmingham. Ph.D.

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Abstract

Transposon-directed insertion-site sequencing (TraDIS) is a high-throughput method that couples transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify the essential or conditionally essential genome of an organism, linking phenotype with genotype. A mini Tn5 transposon library was constructed in the model organism Escherichia coli K-12 strain BW25113. With a median distance between insertions of 3 bp, this is one of the most dense libraries published and provides a valuable tool for whole genome screening. Analysis of this library revealed subtle differences between the TraDIS method and the gold-standard gene-deletion method to determine the essential genome of an organism. This included, but was not limited to, the identification of transposon insertion bias, the boundaries of essential domains within a gene, and short essential domains within intragenic regions. Insertion bias was subsequently found to reveal the position of essential promoters and novel regulatory elements. This library was further used to probe the genomic requirement for survival in the presence of the clinically relevant antibiotic polymyxin B at sub-inhibitory concentrations. Among this data a gene of unknown function, yhcB, was identified and found to have a fundamental role in cell envelope biogenesis. The density of this library enabled identification of previously unreported genomic features. The results presented highlight the potential applications of TraDIS as a tool for a wide range of biological questions.

Type of Work:	Thesis (Doctorates > Ph.D.)
Award Type:	Doctorates > Ph.D.
Supervisor(s):	Supervisor(s) Email ORCID Henderson, Ian R. UNSPECIFIED orcid.org/0000-0002-9954-4977 Lund, Peter A. UNSPECIFIED UNSPECIFIED
Licence:	All rights reserved
College/Faculty:	Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department:	School of Biosciences
Funders:	Biotechnology and Biological Sciences Research Council
Subjects:	Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology
URI:	http://etheses.bham.ac.uk/id/eprint/9187

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