Recently, the functionality of under oil open microfluidics was expanded from droplet-based operations to include lateral flow in under oil aqueous channels. However, the resolution of the under oil fluidic channels reported so far is still far from comparable with that of closed-channel microfluidics (millimeters versus micrometers). Here, enabled by exclusive liquid repellency and an under oil sweep technique, open microchannels can now be prepared under oil (rather than in air), which shrinks the channel dimensions up to three orders of magnitude compared to previously reported techniques. Spatial trapping of different cellular samples and advanced control of mass transport (i.e., enhanced upper limit of flow rate, steady flow with passive pumping, and reversible fluidic valves) were achieved with open-channel designs. We apply these functional advances to enable dynamic measurements of dispersion from a pathogenic fungal biofilm. The ensemble of added capabilities reshapes the potential application space for open microfluidics.
Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control integrated with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization mediated by the proteins CRY2 and CIB1. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light intensity and duty cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This study allows for rapid generation and prototyping of optogenetic circuits to control gene expression in S. cerevisiae.
Gene Expression Regulation, Fungal , Optogenetics/methods , Promoter Regions, Genetic , Saccharomyces cerevisiae/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cloning, Molecular , Cryptochromes/genetics , Cryptochromes/metabolism , Gene Expression Regulation, Fungal/genetics , Gene Expression Regulation, Fungal/radiation effects , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Zinc Fingers/genetics
INTRODUCTION: Controlling gene expression is a fundamental goal of basic and synthetic biology because it allows insight into cellular function and control of cellular activity. We explored the possibility of generating an optogenetic repressor of gene expression in the model organism Saccharomyces cerevisiae by using light to control the nuclear localization of nuclease-dead Cas9, dCas9. METHODS: The dCas9 protein acts as a repressor for a gene of interest when localized to the nucleus in the presence of an appropriate guide RNA (sgRNA). We engineered dCas9, the mammalian transcriptional repressor Mxi1, and an optogenetic tool to control nuclear localization (LINuS) as parts in an existing yeast optogenetic toolkit. This allowed expression cassettes containing novel dCas9 repressor configurations and guide RNAs to be rapidly constructed and integrated into yeast. RESULTS: Our library of repressors displays a range of basal repression without the need for inducers or promoter modification. Populations of cells containing these repressors can be combined to generate a heterogeneous population of yeast with a 100-fold expression range. We find that repression can be dialed modestly in a light dose- and intensity-dependent manner. We used this library to repress expression of the lanosterol 14-alpha-demethylase Erg11, generating yeast with a range of sensitivity to the important antifungal drug fluconazole. CONCLUSIONS: This toolkit will be useful for spatiotemporal perturbation of gene expression in Saccharomyces cerevisiae. Additionally, we believe that the simplicity of our scheme will allow these repressors to be easily modified to control gene expression in medically relevant fungi, such as pathogenic yeasts.
