4 research outputs found
A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae
Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production
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A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae.
Despite the extensive use of Saccharomyces cerevisiae as a platform for synthetic biology, strain engineering remains slow and laborious. Here, we employ CRISPR/Cas9 technology to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility. The toolkit includes 23 Cas9-sgRNA plasmids, 37 promoters of various strengths and temporal expression profiles, and 10 protein-localization, degradation and solubility tags. We facilitated the use of these parts via a web-based tool, that automates the generation of DNA fragments for integration. Our system builds upon existing gene editing methods in the thoroughness with which the parts are standardized and characterized, the types and number of parts available and the ease with which our methodology can be used to perform genetic edits in yeast. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled us to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production
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BioParts-A Biological Parts Search Portal and Updates to the ICE Parts Registry Software Platform.
Capturing, storing, and sharing biological DNA parts data are integral parts of synthetic biology research. Here, we detail updates to the ICE biological parts registry software platform that enable these processes, describe our implementation of the Web of Registries concept using ICE, and establish Bioparts, a search portal for biological parts available in the public domain. The Web of Registries enables standalone ICE installations to securely connect and form a distributed parts database. This distributed database allows users from one registry to query and access plasmid, strain, (DNA) part, plant seed, and protein entry types in other connected registries. Users can also transfer entries from one ICE registry to another or make them publicly accessible. Bioparts, the new search portal, combines the ease and convenience of modern web search engines with the capabilities of bioinformatics search tools such as BLAST. This portal, available at bioparts.org, allows anyone to search for publicly accessible biological part information (e.g., NCBI, iGEM, SynBioHub, Addgene), including parts publicly accessible through ICE Registries. Additionally, the portal offers a REST API that enables third-party applications and tools to access the portal's functionality programmatically