Atlason, Úlfur Ágúst (2022) Experimental evolution of substrate specificity in amino acid transporters. Master's Research Project 2, Biomolecular Sciences.
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Abstract
In yeast, amino acid transport through the cell membrane is done by Yeast Amino Acid Transporters (YAT), a family of membrane transport proteins. These proteins share a conserved structure that has diversified for a wide range of transport rate, substrates and substrate specificity. In order to explore how such diversification evolves, directed evolution can be used to explore how mutations alter those characteristics. Previous studies have used both random mutagenesis and targeted mutations to alter the substrate range of transporters. The OrthoRep system utilises orthologous replication of cytoplasmic plasmids for in vivo mutagenesis of target genes in Saccharomyces cerevisiae. The YAT Agp1 was successfully set up in this system. Two types of selective pressure were applied in parallel, towards the uptake of a new substrate, the non-canonical amino acid citrulline, and against the uptake of three toxic amino acid analogues, 4-fluorophenylalanine, 5-fluorotryptophan and β-chlorolanine. 6 new variants were successfully isolated and characterised, 2 from citrulline selection and 4 from 5-fluorotryptophan selection. Surprisingly all demonstrated the ability to take up citrulline. No improvement was observed in resistance to 5-fluorotryptophan. All variants shared the I334N mutation, bringing to question whether it predates the selection process or plays a significant role in the altered phenotype. Overall the project demonstrates a proof-of-concept of using OrthoRep for directe
| Item Type: | Thesis (Master's Research Project 2) |
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| Supervisor name: | Poolman, B. and Obermaier, S. |
| Degree programme: | Biomolecular Sciences |
| Thesis type: | Master's Research Project 2 |
| Language: | English |
| Date Deposited: | 29 Aug 2022 10:40 |
| Last Modified: | 29 Aug 2022 10:40 |
| URI: | https://fse.studenttheses.ub.rug.nl/id/eprint/28512 |
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