Yeast Tools

Dual gene expression cassette vectors with antibiotic selection marker

pCEV-vector with dual promoter, selection casette and two expression casettes.The pCEV vectors allow over-expression of 2-3 genes from one plasmid in yeast using antibiotic resistance for selection. This is particularly useful for industrial strains that do not have engineered auxotrophies, or for heavily engineered strains that have no auxotrophic markers remaining for selection. Genomic integration is also possible. Read the paper and view Figure here, and get the plasmids here.

Novel yeast promoters for dynamic gene regulation

Bar chart of GFP expression vs. promoter. Shows high activity of SUC2 promoter on sucrose, and low activity on glucose.Promoters characterised for different carbon substrates and for exponential and ethanol phase growth – read about them here

Figure: Sucrose responsive promoters: grow on glucose to increase the biomass then switch to sucrose to initiate gene expression (or repression) – read about the approach and view Figure here, and get the Suc promoter plasmids here.

Quorum sensing modules for cell-density-dependent gene control

Three drawings showing population increase in S. cerviciae and enhanced GFP expression with high population density.Engineered quorum-sensing using pheromone-mediated cell-to-cell communication in Saccharomyces cerevisiae. Used to trigger gene expression (or repression using RNAi) according to cell density. Read more and view Figure here. Get the plasmids here.

Figure: (i) When S. cerevisiae population density is low, there is insufficient pheromone (red dots) present to induce GFP expression (blue S. cerevisiae). (ii) Pheromone becomes more concentrated as the population grows; at sufficient pheromone concentration, GFP expression is triggered (green S. cerevisiae). (iii) at sufficient pheromone concentration, GFP is induced across the whole population.

Engineered RNAi for Saccharomyces cerevisiae

The RNAi system from S. castelli was imported into S. cerevisiae for gene knock-down. We tested it for shikimate pathway engineering linked to a quorum sensing module and in a sucrose-response-repression system.

shik-pher-qs-no-bCircuit topology of engineered S. cerevisiae. Tryptophan-initiated pheromone quorum sensing, the shikimate pathway, the pheromone responsive FUS1J2 promoter and RNAi gene knockout were used to produce high levels of PHBA. See Fig.1b, Williams et al, Metabolic Engineering 29 (2015): 124-134, linked above. Click to enlarge.

rnai-suc-res-repDynamic repression of GFP expression using sucrose mediated RNAi. Expression of a hairpin GFP construct is triggered using SUC2 promoter during growth on sucrose, causing GFP expression to be repressed via RNA interference. See Fig.4b, Williams et al, Microbial Cell Factories (2015) 14:43, linked above. Click to enlarge.