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Transcriptional regulation in yeast


You are all welcome to the master's thesis presentation on transcriptional regulation in yeast by Christos Skrekas

Introduction: Development of cell factories can be challenging. Drainage of precursor metabolites and deregulation of cellular homeostasis are usual when pathways that overproduce a valuable product are introduced. Therefore, robust platform strains with enhanced fluxes towards important precursor molecules have to be developed

Method: We used a combinatorial screening approach in yeast for finding transcription levels of selected genes that contribute to enhanced flux towards cytosolic acetyl-CoA and its direct derivative malonyl-CoA, important precursors of valuable products. A malonyl-CoA biosensor with green fluorescence as output was used for the selection of malonyl-CoA overproducers using fluorescence activated cell sorting (FACS). The candidate genes for screening were found by flux balance analysis (FBA) and different transcription levels of those were screened using CRISPRi, a novel approach for sequence-specific transcriptional regulation. In this approach, artificial transcription modulators are specifically guided by a guide RNA (gRNA). A guide RNA library was constructed for the selected genes, and the library was screened with the malonyl-CoA biosensor to identify gRNAs that contribute to high intracellular pools of malonyl-CoA. Also a potential method of multiplexing gRNAs in a single transcript using Csy4 endonuclease was examined, which could be used for simultaneous screenings of multiple gRNAs.

Results: The gRNA library was screened and sorted in two time points. After three rounds of FACS, gRNA sequencing was performed in colonies resulted from single cells that showed the most extreme fluorescence values. The analysis revealed 14 gRNAs in total, of which 6 were common between the two time points and also showed an expansion over the time. This could indicate a selection of those gRNAs as contributors to high malonyl-CoA levels but their effect has to be proven also in independent experiments. Csy4 multiplexing method also seemed to be a suitable for gRNA multiplexing in a single transcript. Therefore screenings of the effect of simultaneous transcriptional regulation of multiple genes is possible.

Supervisors: Florian David, Raphael Ferreira

Examiner: Anders Blomberg

Föreläsare: Christos Skrekas

Datum: 2017-06-19

Tid: 14:00 - 15:00

Kategorier: Naturvetenskap

Arrangör: Department of Marine Sciences

Plats: 10:an, on the 10th floor of the Chemistry building, entrance Kemigården 4 - Johanneberg campus

Kontaktperson: Anders Blomberg

Sidansvarig: Bo Johannesson|Sidan uppdaterades: 2015-06-30

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Utskriftsdatum: 2017-12-16