A Microfluidic Platform for Screening Gene Expression Dynamics across Yeast Strain Libraries.

The relative ease of genetic manipulation in S. cerevisiae is one of its greatest strengths as a model eukaryotic organism. Researchers have leveraged this quality of the budding yeast to study the effects of a variety of genetic perturbations, such as deletion or overexpression, in a high-throughput manner. This has been accomplished by producing a number of strain libraries that can contain hundreds or even thousands of distinct yeast strains with unique genetic alterations. While these strategies have led to enormous increases in our understanding of the functions and roles that genes play within cells, the techniques used to screen genetically modified libraries of yeast strains typically rely on plate or sequencing-based assays that make it difficult to analyze gene expression changes over time. Microfluidic devices, combined with fluorescence microscopy, can allow gene expression dynamics of different strains to be captured in a continuous culture environment; however, these approaches often have significantly lower throughput compared to traditional techniques. To address these limitations, we have developed a microfluidic platform that uses an array pinning robot to allow for up to 48 different yeast strains to be transferred onto a single device. Here, we detail a validated methodology for constructing and setting up this microfluidic device, starting with the photolithography steps for constructing the wafer, then the soft lithography steps for making polydimethylsiloxane (PDMS) microfluidic devices, and finally the robotic arraying of strains onto the device for experiments. We have applied this device for dynamic screens of a protein aggregation library; however, this methodology has the potential to enable complex and dynamic screens of yeast libraries for a wide range of applications. Key features • Major steps of this protocol require access to specialized equipment (i.e., microfabrication tools typically found in a cleanroom facility and an array pinning robot). • Construction of microfluidic devices with multiple different feature heights using photolithography and soft lithography with PDMS. • Robotic spotting of up to 48 different yeast strains onto microfluidic devices.

Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with challenges to proteostasis.

Chromatin instability and protein homeostasis (proteostasis) stress are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of Saccharomyces cerevisiae, a challenge to proteostasis occurs specifically in the fraction of cells with decreased stability within the ribosomal DNA (rDNA). A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)-binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability induced by inhibition of a conserved lysine deacetylase Sir2. Further, loss of rDNA stability induces age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates contribute to age-induced proteostasis decline and limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis stress and highlight the importance of cell-to-cell variability in aging processes.

Genome-scale transcriptional dynamics and environmental biosensing.

Genome-scale technologies have enabled mapping of the complex molecular networks that govern cellular behavior. An emerging theme in the analyses of these networks is that cells use many layers of regulatory feedback to constantly assess and precisely react to their environment. The importance of complex feedback in controlling the real-time response to external stimuli has led to a need for the next generation of cell-based technologies that enable both the collection and analysis of high-throughput temporal data. Toward this end, we have developed a microfluidic platform capable of monitoring temporal gene expression from over 2,000 promoters. By coupling the "Dynomics" platform with deep neural network (DNN) and associated explainable artificial intelligence (XAI) algorithms, we show how machine learning can be harnessed to assess patterns in transcriptional data on a genome scale and identify which genes contribute to these patterns. Furthermore, we demonstrate the utility of the Dynomics platform as a field-deployable real-time biosensor through prediction of the presence of heavy metals in urban water and mine spill samples, based on the the dynamic transcription profiles of 1,807 unique Escherichia coli promoters.