Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.582411
Title: Creating an orthogonal signalling pathway in S. cerevisiae
Author: Arifin, Khaizurin T.
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2013
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.582411  DOI: Not available
Keywords: QH301 Biology ; QK Botany ; QP Physiology
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