A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica.
Milne N, Tramontin LRR, Borodina I
FEMS Yeast Res. 2019 Sep 26. pii: foz062. doi: 10.1093/femsyr/foz062.
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PubMed
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Article
We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules. All of the plasmids listed on this page are required to carry out the entire protocol.
Notes:
- The protocol can be found in the article listed above.
- The yeast strains have been deposited at Euroscarf.
- If you are interested in ordering all 33 plasmids, please contact help@addgene.org to ask about special pricing.