Modular, Synthetic Boolean Logic Gates Enabled in Saccharomyces cerevisiae through T7 Polymerases/CRISPR dCas9...

Synthetic control of gene expression, whether simply promoter selection or higher-order Boolean-style logic, is an important tool for metabolic engineering and synthetic biology. This work develops a suite of orthogonal T7 RNA polymerase systems capable of exerting AND/OR switchlike control over transcription in the yeastSaccharomyces cerevisiae. When linked with CRISPR dCas9-based regulation systems, more complex circuitry is possible including AND/OR/NAND/NOR style control in response to combinations of extracellular copper and galactose. Additionally, we demonstrate that these T7 system designs are modular and can accommodate alternative stimuli sensing as demonstrated through blue light induction. These designs should greatly reduce the time and labor necessary for developing Boolean gene circuits in yeast with novel applications including metabolic pathway control in the future.