Date of Graduation

5-2017

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Jeffrey A. Lewis

Committee Member

Young M. Kwon

Second Committee Member

David S. McNabb

Keywords

Mutant library, Natural variation, Saccharomyces cerevisiae, Tn5, Tn-Seq, Biological sciences

Abstract

One of the main challenges in biology today is the characterization of millions of genes of unknown function being continuously identified in sequencing studies. Transposon mutagenesis is a technique that has been widely used for annotating gene function and has now been combined with next-generation sequencing (Tn-Seq) to assess mutant fitness on a genome wide basis. However, Tn-Seq approaches are often constrained by laborious library preparation protocols which limit the number of organisms or conditions that can be assessed. Random bar code transposon-site sequencing (RB-TnSeq), is a transposon sequencing technique that streamlines library preparation and increases the throughput of mutant fitness profiling by incorporating random DNA barcodes into Tn5 Transposons. Rb-TnSeq has been successfully used for high throughput mutant fitness assays in diverse bacterial species. However, this technique is yet to be applied to a eukaryotic model organism. The goal of this study is to develop tools that allow the construction of barcoded mutant libraries in saccharomyces cerevisiae and describes methods for producing barcoded mutant libraries using a plasmid based or transposome based approach. These library construction protocols can prove to be powerful tools for studying gene function in S. cerevisiae on a genome wide basis.

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