01whole.pdf (5.36 MB)
Download file

Optimising yeast surface display for identifying cellular targets of natural products

Download (5.36 MB)
thesis
posted on 28.03.2022, 23:17 authored by Ruchi Dhaval Mehta
While natural products have long been a valuable source of biologically active compounds, their cellular targets and modes of action are rarely identified. In this project, yeast surface display (YSD) was investigated as a platform technology to rapidly identify the cellular targets of natural products. YSD involves cloning a cDNA library into baker's yeast (Saccharomyces cerevisiae) such that the encoded foreign proteins are expressed fused to the yeast Aga2 surface receptor. Flow cytometry (FACS) is then used to separate yeast cells displaying proteins capable of binding to a fluorescently tagged probe, thereby allowing the cellular targets of the probe to be identified. In Part 1 of this project, a YSD clone displaying the well-studied human protein FKBP was constructed as a positive control. A fluorescently labelled analogue of FK506 (a known inhibitor of FKBP) was then used to optimise a range of FACS parameters for protein-small molecule interactions, including probe concentration, incubation time/temperature, detergent concentration, washing stringency and FACS binning stringency. In Part 2, a YSD cDNA library was constructed from the model nematode Caenorhabditis elegans. These preliminary studies have laid the groundwork for future YSDstudies to identify the cellular targets of a range of biologically active natural products.

History

Table of Contents

1. Contents -- 2. Abstract -- 3. Introduction -- 4.Construction of a YSD clone displaying human protein FKBP2 as a positive control for FACS optimisation -- 5.Optimisation of flow cytometry parameters -- 6. Validation of Bactrocera tryoni cDNA library -- 7. Construction of Caenorhabditis elegans CDNA library -- 8. Project outputs and future directions -- 9, References -- 10. Supplementary data.

Notes

Theoretical thesis. Bibliography: pages 57-60

Awarding Institution

Macquarie University

Degree Type

Thesis MRes

Degree

MRes, Macquarie University, Faculty of Science and Engineering, Department of Molecular Sciences

Department, Centre or School

Department of Molecular Sciences

Year of Award

2017

Principal Supervisor

Andrew Piggott

Rights

Copyright Ruchi Dhaval Mehta 2017 Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (70 pages : illustrations, map)

Former Identifiers

mq:70783 http://hdl.handle.net/1959.14/1267691