This invention is an expression cassette to reliably control gene expression in yeast.
Several recent studies have developed either promoter libraries or terminator libraries leading to the unexpected finding that terminators, like promoters, have expression-enhancing properties. Current studies only vary one part type and therefore cannot investigate composability- interactions when certain promoters/terminators are used in-conjunction, thus making it impossible to predict the gene expression strength of a new promoter-terminator combination. Furthermore, current libraries are not replicate- meaning they define only one particular part at a given expression strength while requiring the reuse of parts, and consequently, inviting instability into many designs. This invention is a yeast parts library that is compatible with a Type IIS cloning standard to enable rapid adoption and simple application, even for large combinatorial designs.
Traditional cloning techniques are largely inefficient, and variations in the multiple cloning site affect gene expression. Bridging oligos and homologous recombination suffer exponential increases in the number of oligos required for multiple combinatorial genetic designs. Type IIS-based methods are a useful alternative, which may allow large scale combinatorial designs to be assembled rapidly. This invention is an expression cassette (promoter-terminator) library which enables the addition of thousands of new parts for which transcriptional strength is known and predictable. 36 promoters and 26 terminators were selected using guidelines such as limited homology, constitutive expression and having no overlaps with known regulatory elements, ORFs, or centromeres. 124 unique promoter-terminator combinations were constructed and their expression strengths determined. This technology allows the generation of libraries of expression cassettes for large-scale construction.
Applicable to large combinatorial designs
Knowledge of transcriptional part interaction
allows pathway optimization
Genetic designs more stable and easy to clone