Arifin, Khaizurin T. (2013) Creating an orthogonal signalling pathway in S. cerevisiae. PhD thesis, University of Warwick.

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Abstract

The pheromone mating pathway in baker’s yeast Saccharomyces cerevisiae enables
this organism to initiate a developmental response upon detection of a mating
pheromone. Stimulation of the Ste2 receptor by an external trigger molecule in one
of the haploid mating cells of yeast MATa, results in the transcriptional induction of
a subset of yeast genes. The aim of this study is to convert this system into a readily
measurable response, to function as a biosensor. It is crucial for the improved
system to be exclusive from the wild-type response. A successful improved system
should be able to detect other peptides, such as peptide markers in diseases. An invivo
luciferase activity assay utilizing a PFUS1:LUC construct, was developed to
report the activation of the pathway. Deletions of both FAR1 and SST2 genes were
proven to provide an increased signal to noise ratio. As an attempt to decouple Ste2
and its ligand, three α-factor analogues; N3G, E7 and C3G, were found to stimulate
the mating pathway in decreasing order. A modified Ste12 (Ste12mod) transcription
factor coupled with a modified pheromone response element (PREmod2)
partnership was also designed. A library of Ste12mod was constructed by random
mutagenesis of the N-terminal DNA-binding domain. The mutant Ste12 plasmids
were transformed into a far1 sst2 ste12 strain complemented with
pFUS1(mod2)K. Screening for mutants with an increased resistance to G418
resulted in 17 unique mutations. Further screening by mating and luciferase assays
narrowed down to one mutation (F45I) that responds to modified PRE. However,
this strain successfully mated with the wild-type, which warrant further
modifications and screening to achieve a full orthogonal system. The results in this
study not only provided more insight to the physical interaction between Ste12 and
PRE, but also the possibility of uncoupling a pathway in an already developed
system.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology Q Science > QK Botany Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Saccharomyces cerevisiae -- Physiology, Pheromones -- Receptors, Mutation (Biology), Systems biology, Biological systems, Cellular signal transduction
Official Date:	March 2013
Dates:	Date Event March 2013 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	McCarthy, John E. G.
Extent:	xiii, 143 leaves : illustrations.
Language:	eng

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