Can transposon directed insertion-site sequencing be used to predict possible outcomes of evolution?

Milner, Mathew Thomas (2022). Can transposon directed insertion-site sequencing be used to predict possible outcomes of evolution? University of Birmingham. Ph.D.

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Laboratory-based evolution has become a tool that is widely used to understand an organism’s response to stressful environments through linking the genotype to the phenotype. Within laboratory evolution, the role that loss of function mutations play in adaptation is a topic of debate, with recent observations suggesting that adaptive loss of function mutations are a common adaptive strategy. One limiting factor of this technique is that the time taken to conduct a single experiment can be extensive. With these points in mind, we proposed to see if a short term selection experiment on a high density transposon library, using Transposon Directed Insertion-site Sequencing (TraDIS) to analyse the data, would produce results which correlate with those from long-term evolution experiments. Since TraDIS provides a measure of relative contributions to fitness of each gene, in principle it should be possible to use TraDIS to identify genes whose loss of function provides a fitness benefit on a significantly shorter timescale. Previously in our laboratory, five populations of E. coli K-12 MG1655 were evolved in a dynamic pH environment by daily passaging over five months in unbuffered LB, starting at pH 4.5. Whole genome resequencing of the final populations and clones revealed many striking similarities in the evolutionary trajectories of these populations. Therefore, to explore the hypothesis that short term selection of a high density transposon library could identify genes that were also found in the five month evolution experiment, an E. coli K-12 MG1655 transposon library was constructed and passaged for 10 days under similar conditions as the evolution experiment at both pH 4.5 and pH 7. TraDIS analysis showed that, within these populations, insertions in a few genes had accumulated, suggesting there was a fitness advantage for a strain carrying these insertions. These genes showed a significant overlap with the ones identified in the evolution experiment. These results highlight a possible alternative approach to laboratory evolution when attempting to understand an organism’s response to stress, providing a foundation for future work to explore different conditions.

Research data supporting this thesis can be found on the University of Birmingham eData repository at:

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
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 > Q Science (General)
Q Science > QH Natural history
Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Q Science > QR Microbiology


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