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Exploring the genome rearrangement system, SCRaMbLE, to introduce DNA into Saccharomyces cerevisiae

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thesis
posted on 28.03.2022, 11:36 by Elizabeth Louise Izolde Wightman
A novel genome rearrangement tool, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP mediated evolution), has been designed for the synthetic genome of S. cerevisiae. Collaborators of the 'yeast 2.0' project initially construct one version of the synthetic genome. However, SCRaMbLE can rapidly generate billions of unique genomes in even a small culture. Only one of the 16 chromosomes is currently complete, limiting full genome rearrangement by SCRaMbLE. As a contribution to the international project, I constructed and integrated a 'megachunk' of synthetic chromosome 14 into S. cerevisiae. Currently, SCRaMbLE is limited to S. cerevisiae DNA only. I aimed to explore SCRaMbLE in a novel application: as a tool to introduce foreign DNA into S. cervisiae. To develop protocols, the S. cerevisiae marker gene URA3 was used to SCRaMbLE into yeast. SCRaMbLEing URA3 increasing the transformation efficiency significantly compared to non-SCRaMbLEd URA3.gDNA from the filamentous fungus Trichoderma reesei was used as an example of SCRaMbLEing large libraries of foreign DNA into yeast. Successful attachment of SCRaMbLE recombination sites to gDNA fragments was achieved using a cloning approach, gDNA fragments were SCRaMbLEd into yeast and cellulase activity was screened.

History

Alternative Title

SCRaMbLEing DNA into saccharomyces cerevisiae.

Table of Contents

Chapter 1. Introduction -- Chapter 2. Construction of megachunk O of yeast synthetic chromosome XIV -- Chapter 3. Proof of concept : SCRaMbLEing the URA3 marker into yeast -- Chapter 4. PCR approaches to attach recombination sequences to T. reesei DNA -- Chapter 5. A cloning approach of loxP attachment to T. reesei DNA and subsequent SCRaMbLEing into S. cerevisiae -- Chapter 6. Summary, conclusions and future direction.

Notes

Bibliography: pages 56-59 Empirical thesis.

Awarding Institution

Macquarie University

Degree Type

Thesis MRes

Degree

MRes, Macquarie University, Faculty of Science and Engineering, Department of Chemistry and Biomolecular Sciences

Department, Centre or School

Department of Chemistry and Biomolecular Sciences

Year of Award

2015

Principal Supervisor

Helena Nevalainen

Rights

Copyright Elizabeth Louise Izolde Wightman 2015. Copyright disclaimer: http://www.copyright.mq.edu.au

Language

English

Extent

1 online resource (viii, 63 pages) illustrations (some colour)

Former Identifiers

mq:47053 http://hdl.handle.net/1959.14/1089501