Chapter title |
Coupling Yeast Golden Gate and VEGAS for Efficient Assembly of the Violacein Pathway in Saccharomyces cerevisiae
|
---|---|
Chapter number | 14 |
Book title |
Synthetic Metabolic Pathways
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7295-1_14 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7294-4, 978-1-4939-7295-1
|
Authors |
James Chuang, Jef D. Boeke, Leslie A. Mitchell |
Abstract |
The ability to express non-native pathways in genetically tractable model systems is important for fields such as synthetic biology, genetics, and metabolic engineering. Here we describe a modular and hierarchical strategy to assemble multigene pathways for expression in S. cerevisiae. First, discrete promoter, coding sequence, and terminator parts are assembled in vitro into Transcription Units (TUs) flanked by adapter sequences using "yeast Golden Gate" (yGG), a type IIS restriction enzyme-dependent cloning strategy. Next, harnessing the natural capacity of S. cerevisiae for homologous recombination, TUs are assembled into pathways and expressed using the "Versatile Genetic Assembly System" (VEGAS) in yeast. Coupling transcription units constructed by yGG with VEGAS assembly is a generic and flexible workflow to achieve pathway expression in S. cerevisiae. This protocol describes assembly of a five TU pathway for yeast production of violacein, a pigment derived from Chromobacterium violaceum. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 1 | 100% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 41 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 8 | 20% |
Researcher | 8 | 20% |
Student > Master | 6 | 15% |
Student > Bachelor | 3 | 7% |
Professor > Associate Professor | 3 | 7% |
Other | 2 | 5% |
Unknown | 11 | 27% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 14 | 34% |
Agricultural and Biological Sciences | 7 | 17% |
Chemical Engineering | 2 | 5% |
Pharmacology, Toxicology and Pharmaceutical Science | 1 | 2% |
Environmental Science | 1 | 2% |
Other | 2 | 5% |
Unknown | 14 | 34% |