Parallel profiling of mutants for chronological lifespan
Lead Research Organisation:
University College London
Department Name: Structural Molecular Biology
Abstract
Strategic Research Priority: World Class Underpinning Bioscience
Understanding the main players involved in cellular senescence is key to uncovering the mechanisms required for the promotion of healthy ageing. Fission yeast and budding yeast are distantly related organisms, both of which are exceptional model organisms used for ageing studies. The two yeasts therefore offer complementary insights into the ageing research. In this project, we employ barcode sequencing to S. pombe genome-wide deletion library of nonessential genes, accounting for more than 80% of the genome, in order to aid the discovery of mutants with long and short lifespans under different environmental conditions. We anticipate further utilization of these mutants for drug screen experiments. The barcodes are a unique 20-nucleotide sequence that laterally spans the KanMX4 cassette, which is inserted in the region of each of the deleted gene, and thus, the barcodes act as molecular marks with direct quantitation of the mutants during chronological assays. The mutant counts is captured by PCR and highlighted in the sequencing data extrapolated from DNA libraries.
Methods that direct the project include, but not limited to, the setting and optimization of the barcode sequencing technique, DNA library preparation, fluorescence microscopy for cellular imaging along with FACS analysis.
Understanding the main players involved in cellular senescence is key to uncovering the mechanisms required for the promotion of healthy ageing. Fission yeast and budding yeast are distantly related organisms, both of which are exceptional model organisms used for ageing studies. The two yeasts therefore offer complementary insights into the ageing research. In this project, we employ barcode sequencing to S. pombe genome-wide deletion library of nonessential genes, accounting for more than 80% of the genome, in order to aid the discovery of mutants with long and short lifespans under different environmental conditions. We anticipate further utilization of these mutants for drug screen experiments. The barcodes are a unique 20-nucleotide sequence that laterally spans the KanMX4 cassette, which is inserted in the region of each of the deleted gene, and thus, the barcodes act as molecular marks with direct quantitation of the mutants during chronological assays. The mutant counts is captured by PCR and highlighted in the sequencing data extrapolated from DNA libraries.
Methods that direct the project include, but not limited to, the setting and optimization of the barcode sequencing technique, DNA library preparation, fluorescence microscopy for cellular imaging along with FACS analysis.
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
| 1618819 | Studentship | BB/M009513/1 | 01/10/2015 | 30/09/2019 | Catalina Romila |
| Description | Improved characterisation of the fission yeast latest deletion collection. An attractive technique, Bar-Seq was successfully developed and validated and can be used as a high throughput method to answer genetics questions. Following our high-throughput experiment analysis we uncovered several novel longevity associated genes in yeast, and these have been independently validated. |
| Exploitation Route | The characterised fission yeast deletion collection can be used to carry out genetics testing of any kind, which together with Bar-Seq can be used as a high-throughput method for the community. Bar-Seq has applications even outside of the fission yeast community. |
| Sectors | Education,Healthcare,Other |
| URL | https://github.com/Catalina37/catalina.github.io |
| Description | Characterisation of the fission yeast Bioneer collection for use in further academic research studies and throughout my PhD. The Bar-Seq method was a success developed during my PhD and will be used in the lab extensively to answer genetics studies. |
| First Year Of Impact | 2019 |
| Sector | Other |