Investigation of Ty1 sites for multi-copy integration of a Brazzein expression system using CRISPR/CAS9 in Saccharomyces cerevisiae

Ellinghaus, Alric 2019, Investigation of Ty1 sites for multi-copy integration of a Brazzein expression system using CRISPR/CAS9 in Saccharomyces cerevisiae, B.Science (Hons) thesis, School of Life and Environmental Sciences, Deakin University.

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Title Investigation of Ty1 sites for multi-copy integration of a Brazzein expression system using CRISPR/CAS9 in Saccharomyces cerevisiae
Author Ellinghaus, Alric
Institution Deakin University
School School of Life and Environmental Sciences
Faculty Faculty of Science, Engineering and Built Environment
Degree type Honours
Degree name B.Science (Hons)
Thesis advisor Dichtl, BernhardORCID iD for Dichtl, Bernhard orcid.org/0000-0001-5514-4982
Date submitted 2019-11-08
Keyword(s) CRISPR
brazzein
yeast
gene expression
Summary Obesity and its comorbidities have become a global concern as a result of widespread climbing obesity rates. Sugar, particularly sucrose is thought to play a dominant causative role in the observed trends, and many believe reducing sucrose intake to be a key solution. A new generation of protein-based sweeteners have been isolated from fruits of plants native to tropical climates. One of these sweet-tasting proteins, brazzein, is a promising alternative sweetener. The aim of this project is to utilize recent advances in recombinant DNA technology and yeast engineering to construct a Saccharomyces cerevisiae expression system capable of producing high titres of the brazzein protein.
Results: A gene cassette was constructed with the PTEF1 promoter, an ORF encoding an αMF secretory signal fused to the N-terminus of brazzein and the TCYC1 terminator.
Brazzein expression cassettes were integrated at varying copy number into Ty1-LTRs in Saccharomyces cerevisiae via a CRISPR/Cas9 mediated approach. Quantitative PCR demonstrated that one strain (DVT20) was estimated to contain approximately 6 copies of the brazzein cassette. Tricine-SDS PAGE analysis showed a protein whose levels increased with copy number but was larger than the expected size of brazzein. The protein may be brazzein with an un-cleaved αMF secretory peptide.
Conclusion: CRISPR/Cas9 was used to successfully used to integrate multiple copies of brazzein at Ty1 LTRs.
Language eng
Indigenous content off
Field of Research 0601 Biochemistry and Cell Biology
Description of original 102 p.
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Persistent URL http://hdl.handle.net/10536/DRO/DU:30133314

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