Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

Leslie A. Mitchell, James Chuang, Neta Agmon, Chachrit Khunsriraksakul, Nick A. Phillips, Yizhi Cai, David M. Truong, Ashan Veerakumar, Yuxuan Wang, María Mayorga, Paul Blomquist, Praneeth Sadda, Joshua Trueheart, Jef D. Boeke

Research output: Contribution to journalArticle

Abstract

We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.

Original languageEnglish (US)
Pages (from-to)6620-6630
Number of pages11
JournalNucleic Acids Research
Volume43
Issue number13
DOIs
StatePublished - Apr 27 2015

Fingerprint

Saccharomyces cerevisiae
Yeasts
Genes
Workflow
Homologous Recombination
Carotenoids
Genome

ASJC Scopus subject areas

  • Genetics

Cite this

Mitchell, L. A., Chuang, J., Agmon, N., Khunsriraksakul, C., Phillips, N. A., Cai, Y., ... Boeke, J. D. (2015). Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic Acids Research, 43(13), 6620-6630. https://doi.org/10.1093/nar/gkv466

Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. / Mitchell, Leslie A.; Chuang, James; Agmon, Neta; Khunsriraksakul, Chachrit; Phillips, Nick A.; Cai, Yizhi; Truong, David M.; Veerakumar, Ashan; Wang, Yuxuan; Mayorga, María; Blomquist, Paul; Sadda, Praneeth; Trueheart, Joshua; Boeke, Jef D.

In: Nucleic Acids Research, Vol. 43, No. 13, 27.04.2015, p. 6620-6630.

Research output: Contribution to journalArticle

Mitchell, LA, Chuang, J, Agmon, N, Khunsriraksakul, C, Phillips, NA, Cai, Y, Truong, DM, Veerakumar, A, Wang, Y, Mayorga, M, Blomquist, P, Sadda, P, Trueheart, J & Boeke, JD 2015, 'Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae', Nucleic Acids Research, vol. 43, no. 13, pp. 6620-6630. https://doi.org/10.1093/nar/gkv466
Mitchell LA, Chuang J, Agmon N, Khunsriraksakul C, Phillips NA, Cai Y et al. Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic Acids Research. 2015 Apr 27;43(13):6620-6630. https://doi.org/10.1093/nar/gkv466
Mitchell, Leslie A. ; Chuang, James ; Agmon, Neta ; Khunsriraksakul, Chachrit ; Phillips, Nick A. ; Cai, Yizhi ; Truong, David M. ; Veerakumar, Ashan ; Wang, Yuxuan ; Mayorga, María ; Blomquist, Paul ; Sadda, Praneeth ; Trueheart, Joshua ; Boeke, Jef D. / Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. In: Nucleic Acids Research. 2015 ; Vol. 43, No. 13. pp. 6620-6630.
@article{e5dcf77d502e43df9d4bf98a8f02db3e,
title = "Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae",
abstract = "We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.",
author = "Mitchell, {Leslie A.} and James Chuang and Neta Agmon and Chachrit Khunsriraksakul and Phillips, {Nick A.} and Yizhi Cai and Truong, {David M.} and Ashan Veerakumar and Yuxuan Wang and Mar{\'i}a Mayorga and Paul Blomquist and Praneeth Sadda and Joshua Trueheart and Boeke, {Jef D.}",
year = "2015",
month = "4",
day = "27",
doi = "10.1093/nar/gkv466",
language = "English (US)",
volume = "43",
pages = "6620--6630",
journal = "Nucleic Acids Research",
issn = "1362-4962",
publisher = "Oxford University Press",
number = "13",

}

TY - JOUR

T1 - Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae

AU - Mitchell, Leslie A.

AU - Chuang, James

AU - Agmon, Neta

AU - Khunsriraksakul, Chachrit

AU - Phillips, Nick A.

AU - Cai, Yizhi

AU - Truong, David M.

AU - Veerakumar, Ashan

AU - Wang, Yuxuan

AU - Mayorga, María

AU - Blomquist, Paul

AU - Sadda, Praneeth

AU - Trueheart, Joshua

AU - Boeke, Jef D.

PY - 2015/4/27

Y1 - 2015/4/27

N2 - We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.

AB - We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by 'VEGAS adapter' (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.

UR - http://www.scopus.com/inward/record.url?scp=84939572965&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84939572965&partnerID=8YFLogxK

U2 - 10.1093/nar/gkv466

DO - 10.1093/nar/gkv466

M3 - Article

C2 - 25956652

AN - SCOPUS:84939572965

VL - 43

SP - 6620

EP - 6630

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 1362-4962

IS - 13

ER -