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Isolation of a strong constitutive promoter from Euglena gracilis

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posted on 28.03.2022, 11:36 authored by Bishal Khatiwada
Euglena gracilis is a unicellular protist currently used to produce nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organisms requires the development of genetic engineering tools. Chloroplast transformation using the non-native promoter (CaMV 35S) has been reported, but there is no published information about the use of native promoters for recombinant gene expression in Euglena. The aim of this research was to isolate a strong constitutive promoter with a view to establish an expression vector for E. gracilis. Proteins produced during dark cultivation were separated using 2D SDS-PAGE and identified by mass spectrometry. One of the highly expressed proteins was glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The gapC gene encoding GAPDH was identified using BLAST. The 5’ UTR region of the gene was amplified by PCR using gDNA of E. gracilis as a template and inserted into a promoterless E. coli and yeast plasmids containing a reporter gene (β-gal or eGFP). Promoter activity was verified by successful expression of EGFP in yeast. Sequence analysis of the putative Euglena promoter revealed the presence of eukaryotic promoter elements like CAAT box, GC Box and myb-like transcriptional activator. Final confirmation of the promoter activity will be carried out in Euglena after a suitable transformation method has been established.

History

Table of Contents

Chapter 1. Introduction -- Chapter 2. Materials and methods -- Chapter 3. Results -- Chapter 4. Discussion -- Chapter 5. Conclusions and future directions -- References -- Appendices.

Notes

Bibliography: pages 51-57 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

2016

Principal Supervisor

Helena Nevalainen

Additional Supervisor 1

Angela Sun

Rights

Copyright Bishal Khatiwada 2016. Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (x, 68 pages) colour illustrations

Former Identifiers

mq:69295 http://hdl.handle.net/1959.14/1252897