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.
(Link opens in a new window)
PubMed
(Link opens in a new window)
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 [email protected] to ask about special pricing.
Plasmids from the Teaching Protocol
| ID | Plasmid | Description | |
|---|---|---|---|
| 73273 | pCfB3035 | (Empty Backbone) EasyClone-MarkerFree backbone vector for the addition of 1 or 2 genes and promoters for integration into site X-4 (Chr X: 236336..237310) | Add to Cart |
| 73276 | pCfB2904 | (Empty Backbone) EasyClone-MarkerFree backbone vector for the addition of 1 or 2 genes and promoters for integration into site XI-3 (Chr XI: 93378..94567) | Add to Cart |
| 73281 | pCfB2909 | (Empty Backbone) EasyClone-MarkerFree backbone vector for the addition of 1 or 2 genes and promoters for integration into site XII-5 (Chr XII: 839226..840357) | Add to Cart |
| 73294 | pCfB3052 | EasyClone-MarkerFree guiding RNA vector to direct Cas9 to cut at sites X-4, XI-3, and XII-5 | Add to Cart |
| 78230 | p1977 | Example plasmid containing a bidirectional promoter of pTDH3 and pTEF1 | Add to Cart |
| 78231 | pCfB2312 | Expresses Cas9 protein, under the control of the TEF1 promoter, and CYC terminator. KanMX resistance | Add to Cart |
| 78232 | pTAJAK-71 | (Empty Backbone) Empty vector, for the insertion of gRNA expression cassettes into the vector. | Add to Cart |
| 106151 | pCfB6364 | EasyCloneYALI system-based yeast integrative vector expressing Cas9 carrying D-serine marker, integration into Yarrowia lipolytica ku70 locus, amp resistance | Add to Cart |
| 106152 | pCfB6682 | (Empty Backbone) EasyCloneYALI system-based yeast integrative vector for markerfree integration via CRISPR/Cas9 to be used in combination with pCfB6627, integration into Yarrowia lipolytica chromosomal location IntC_2, USER site for cloning, amp resistance | Add to Cart |
| 106154 | pCfB6684 | (Empty Backbone) EasyCloneYALI system-based yeast integrative vector for markerfree integration via CRISPR/Cas9 to be used in combination with pCfB6631, integration into Yarrowia lipolytica chromosomal location IntD_1, USER site for cloning, amp resistance | Add to Cart |
| 106155 | pCfB6677 | (Empty Backbone) EasyCloneYALI system-based yeast integrative vector for markerfree integration via CRISPR/Cas9 to be used in combination with pCfB6632, integration into Yarrowia lipolytica chromosomal location IntE_1, USER site for cloning, amp resistance | Add to Cart |
| 106159 | pCfB6627 | EasyCloneYALI system-based yeast gRNA expression vector carrying a nourseothricin-resistance marker, helps to integrate vector pCfB6682 into Yarrowia lipolytica chromosomal location IntC_2, amp resistance | Add to Cart |
| 106161 | pCfB6631 | EasyCloneYALI system-based yeast gRNA expression vector carrying a nourseothricin-resistance marker, helps to integrate vector pCfB6684 into Yarrowia lipolytica chromosomal location IntD_1, amp resistance | Add to Cart |
| 106162 | pCfB6633 | EasyCloneYALI system-based yeast gRNA expression vector carrying a nourseothricin-resistance marker, helps to integrate vector pCfB6677 into Yarrowia lipolytica chromosomal location IntE_1, amp resistance | Add to Cart |
| 106166 | pCfB3405 | (Empty Backbone) EasyCloneYALI system-based yeast episomal vector carrying a nourseothricin-resistance marker for Yarrowia lipolytica, can be used for gRNA expression, amp resistance | Add to Cart |
| 126905 | pCfB8739 | Template for PCR amplification of XdCrtYB | Add to Cart |
| 126906 | pCfB8740 | Template for PCR amplification of XdCrtI | Add to Cart |
| 126907 | pCfB8741 | Template for PCR amplification of XdCrtE | Add to Cart |
| 126908 | pCfB8742 | Template for PCR amplification of pGPD1-pFBA1 double promoter biobrick for Yarrowia lipolytica | Add to Cart |
| 126909 | pCfB8818 | Template for PCR amplification of HMG1from Yarrowia lipolytica | Add to Cart |
| 126910 | pCfB8792 | Template for PCR amplification of ADE2 gRNA biobrick for targeting Saccharomyces cerevisiae ADE2 gene | Add to Cart |
| 126911 | pCfB5191 | Plasmid containing gRNA expression cassettes targeting the X-4 and XII-5 integration sites in Saccharomyces cerevisiae | Add to Cart |
| 126912 | pCfB8622 | Plasmid containing gRNA expression cassette targeting ADE2 in Saccharomyces cerevisiae | Add to Cart |
| 126913 | pCfB8379 | Plasmid containing integration cassette at X-4 integration site in Saccharomyces cerevisiae for overexpression of XdCrtI and XdCrtYB | Add to Cart |
| 126914 | pCfB8380 | Plasmid containing integration cassette at XII-5 integration site in Saccharomyces cerevisiae for overexpression of XdCrtE | Add to Cart |
| 126915 | pCfB8381 | Plasmid containing integration cassette at XI-3 integration site in Saccharomyces cerevisiae for overexpression of XdCrtI and tHMG1 | Add to Cart |
| 126916 | pCfB8382 | Plasmid containing integration cassette at XI-3 integration site in Saccharomyces cerevisiae for overexpression of XdCrtI | Add to Cart |
| 126917 | pCfB8383 | Plasmid containing integration cassette at XI-3 integration site in Saccharomyces cerevisiae for overexpression of tHMG1 | Add to Cart |
| 126918 | pCfB8748 | Plasmid containing integration cassette at IntE1 integration site in Yarrowia lipolytica for overexpression of XdCrtE | Add to Cart |
| 126919 | pCfB8644 | Plasmid containing integration cassette at IntC2 integration site in Yarrowia lipolytica for overexpression of XdCrtI and XdCrtYB | Add to Cart |
| 126920 | pCfB8786 | Plasmid containing integration cassette at IntD1 integration site in Yarrowia lipolytica for overexpression of XdCrtI | Add to Cart |
| 126921 | pCfB8787 | Plasmid containing integration cassette at IntD1 integration site in Yarrowia lipolytica for overexpression of HMG1 | Add to Cart |
| 126922 | pCfB8788 | Plasmid containing integration cassette at IntD1 integration site in Yarrowia lipolytica for overexpression of XdCrtI and HMG1 | Add to Cart |
Note: the plasmids listed below were deposited with the article, but they are more fully described in the complete table above.
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 126905 | pCfB8739 | Template for PCR amplification of XdCrtYB |
| 126906 | pCfB8740 | Template for PCR amplification of XdCrtI |
| 126907 | pCfB8741 | Template for PCR amplification of XdCrtE |
| 126908 | pCfB8742 | Template for PCR amplification of pGPD1-pFBA1 double promoter biobrick for Yarrowia lipolytica |
| 126909 | pCfB8818 | Template for PCR amplification of HMG1from Yarrowia lipolytica |
| 126910 | pCfB8792 | Template for PCR amplification of ADE2 gRNA biobrick for targeting Saccharomyces cerevisiae ADE2 gene |
| 126911 | pCfB5191 | Plasmid containing gRNA expression cassettes targeting the X-4 and XII-5 integration sites in Saccharomyces cerevisiae |
| 126912 | pCfB8622 | Plasmid containing gRNA expression cassette targeting ADE2 in Saccharomyces cerevisiae |
| 126913 | pCfB8379 | Plasmid containing integration cassette at X-4 integration site in Saccharomyces cerevisiae for overexpression of XdCrtI and XdCrtYB |
| 126914 | pCfB8380 | Plasmid containing integration cassette at XII-5 integration site in Saccharomyces cerevisiae for overexpression of XdCrtE |
| 126915 | pCfB8381 | Plasmid containing integration cassette at XI-3 integration site in Saccharomyces cerevisiae for overexpression of XdCrtI and tHMG1 |
| 126916 | pCfB8382 | Plasmid containing integration cassette at XI-3 integration site in Saccharomyces cerevisiae for overexpression of XdCrtI |
| 126917 | pCfB8383 | Plasmid containing integration cassette at XI-3 integration site in Saccharomyces cerevisiae for overexpression of tHMG1 |
| 126918 | pCfB8748 | Plasmid containing integration cassette at IntE1 integration site in Yarrowia lipolytica for overexpression of XdCrtE |
| 126919 | pCfB8644 | Plasmid containing integration cassette at IntC2 integration site in Yarrowia lipolytica for overexpression of XdCrtI and XdCrtYB |
| 126920 | pCfB8786 | Plasmid containing integration cassette at IntD1 integration site in Yarrowia lipolytica for overexpression of XdCrtI |
| 126921 | pCfB8787 | Plasmid containing integration cassette at IntD1 integration site in Yarrowia lipolytica for overexpression of HMG1 |
| 126922 | pCfB8788 | Plasmid containing integration cassette at IntD1 integration site in Yarrowia lipolytica for overexpression of XdCrtI and HMG1 |