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Project properties |
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| Title | Orthogonal DNA replication in yeast based on poxviral machinery |
| Group | Systems and Synthetic Biology |
| Project type | thesis |
| Credits | 36 |
| Supervisor(s) | Stefan Hoffmann |
| Examiner(s) | Stefan Hoffmann, Rob Smith |
| Contact info | Robert1.smith@wur.nl |
| Begin date | 2025/02/21 |
| End date | |
| Description | Poxviruses are animal viruses that possess double-stranded linear DNA genomes with covalently closed hairpin termini, and infected cells are known to also replicate circular DNA without specific replication signals. Unusual for the lifecycle of DNA viruses, DNA replication and transcription of poxviruses occur entirely in the cytoplasm through their own molecular machinery encoded in their genomes.
We aim to create a system for replicating DNA within yeast cells using molecular machinery of poxviruses. Similar to a virtual machine on a computer running independently of the main operating system, this DNA replication system will function separately from the yeast's natural processes, taking place in the cytoplasm instead of the nucleus. This technology has exciting and diverse potential applications. We envision its eventual use to broaden the genetic alphabet and to create genetic devices that work in different species, opening up new possibilities for biotechnology and genetic engineering. The first aim is to assess completeness of poxviral DNA replication genes for replication of a circular DNA in yeast. Required genes are being taken from vaccinia virus, which is the prototypical poxvirus and has a well-annotated genome. Their expression in yeast will be confirmed, and optimised if required, and all genes will be assembled into a single construct. 11 poxviral genes are thought to be essential for DNA replication, 6 of which could already be expressed at easily detectable levels in yeast when tested with a single strong promoter. Different promoters will be tested to achieve detectable expression for each of the genes. Thanks to the use of a modular cloning standard, assemblies with different promoters and terminators as well as higher-order assemblies can readily be made. Some proteins might need to be co-expressed, if they are unstable in the absence of their interaction partners, as is likely the case for E9, A20 and D4. References related to project: 10.1101/cshperspect.a010199, 10.1074/jbc.M511239200, 10.1093/nar/gkv464 |
| Used skills | |
| Requirements | Knowledge of PCR and molecular cloning, experience with Golden Gate cloning in particular and Western blotting is desirable. |