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Figure 1 | Microbial Cell Factories

Figure 1

From: Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae

Figure 1

Assembly of eYACs. (a) Genes of interest are cloned into Entry vectors and amplified in E. coli. (b) Expression cassettes (hatched and white) containing the genes are generated by a double digest of Entry vectors, leaving cassettes with compatible sticky ends, while backbone DNA (gray) is left with blunt ends. (c) Cassettes are concatenated by ligation. In parallel, YAC arms (checked), supplying all necessary elements for chromosomal function, are prepared by double digest of a pYAC4 derivative. Both long and short arms have compatible, sticky overhangs at one end and telomeres at the other. The long arm also contains a centromere (checked oval) and an autonomous replicating sequence (ARS). Each arm also carries an auxotrophic selection marker (long arm TRP1 and short arm LEU2). (d) Arms are ligated to the ends of cassette concatemers to form eYACs.

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Microbial Cell Factories

ISSN: 1475-2859

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