Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae

James Chuang, Jef D. Boeke, Leslie A. Mitchell

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

LanguageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages211-225
Number of pages15
Volume1671
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1671
ISSN (Print)1064-3745

Fingerprint

Saccharomyces cerevisiae
Yeasts
Genetic Terminator Regions
Chromobacterium
Synthetic Biology
Metabolic Engineering
Genetic Engineering
Workflow
Homologous Recombination
Organism Cloning
violacein
Enzymes

Keywords

  • Hierarchical DNA assembly
  • Metabolic engineering
  • Saccharomyces cerevisiae
  • Versatile Genetic Assembly System (VEGAS)
  • Violacein
  • Yeast Golden Gate (yGG)

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Chuang, J., Boeke, J. D., & Mitchell, L. A. (2018). Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae. In Methods in Molecular Biology (Vol. 1671, pp. 211-225). (Methods in Molecular Biology; Vol. 1671). Humana Press Inc.. DOI: 10.1007/978-1-4939-7295-1_14

Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae. / Chuang, James; Boeke, Jef D.; Mitchell, Leslie A.

Methods in Molecular Biology. Vol. 1671 Humana Press Inc., 2018. p. 211-225 (Methods in Molecular Biology; Vol. 1671).

Research output: Chapter in Book/Report/Conference proceedingChapter

Chuang, J, Boeke, JD & Mitchell, LA 2018, Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae. in Methods in Molecular Biology. vol. 1671, Methods in Molecular Biology, vol. 1671, Humana Press Inc., pp. 211-225. DOI: 10.1007/978-1-4939-7295-1_14
Chuang J, Boeke JD, Mitchell LA. Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae. In Methods in Molecular Biology. Vol. 1671. Humana Press Inc.2018. p. 211-225. (Methods in Molecular Biology). Available from, DOI: 10.1007/978-1-4939-7295-1_14
Chuang, James ; Boeke, Jef D. ; Mitchell, Leslie A./ Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae. Methods in Molecular Biology. Vol. 1671 Humana Press Inc., 2018. pp. 211-225 (Methods in Molecular Biology).
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