A Comprehensive Yeast Tool Kit with Novel Synthetic Regulation Systems for Gene Circuit Construction and Delivery

Complex sophisticated behavior within cells manifests from multiple regulatory networks, in which transcriptional factors (TFs) regulate gene expression, while binding to their cognate operator sequences. Here, we present a framework for building gene circuits and present a set of well-characterized DNA parts for use in Saccharomyces cerevisiae. For the assembly of novel gene circuits, we used Gateway recombination and Gibson Assembly methods; hierarchical assembly of gene circuits comprising multiple transcriptional units was mediated through unique 45 bp sequences. The entire procedure of building a gene circuit from more than 10 basic parts took less than 5 days with only a workload of 1-3 hours per day. Characterization of various promoters and the rules of network design were evaluated by analyzing fluorescent protein expression (GFP and YFP) via flow cytometry. We also characterized a diverse set of promoters consisting of constitutive promoters, native inducible promoters, synthetic inducible promoters, synthetic promoters regulated by activators, and synthetic promoters regulated by repressors. Promoter expression was measured in molecules of equivalent fluorescein (MEFL) units with the following results: (1) constitutive promoters demonstrated a wide range of strengths (up to a 100-fold difference), (2) the new inducible systems enabled an 11-fold change in expression, and (3) the activators/repressors showed a maximum 35-fold and 45-fold change of expression, respectively. In conclusion, this study demonstrates the feasibility of quickly and easily constructing gene circuits for delivery into S. cerevisiae; the utility of a fully characterized set of diverse promoters, activators, and repressors; and the applicability of this system in constructing large-scale gene circuit libraries with reliable gene expression and designing logic operations for a complex network in S. cerevisiae.