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Hotz, M.

Publications and source records attributed to Hotz, M..

2 recordsLinked to original sources

The Yeast Lifespan Machine: a microfluidic platform for automated replicative lifespan measurements

The budding yeast, Saccharomyces cerevisiae, has emerged as a model system for studying the aging processes in eukaryotic cells. However, the full complement of tools available in this organism has not been fully applied, in part because of limitations in throughput that restrict the ability to carry out detailed analyses. Recent advances in microfluidics have provided direct longitudinal observation of entire yeast lifespans, but have not yet achieved the normal scale of operation possible in this model system. Here we present a microfluidic platform, called the Yeast Lifespan Machine, where we combine improvements in microfluidics, image acquisition, and image analysis tools to increase robustness and throughput of lifespan measurements in aging yeast cells. We demonstrate the platforms ability to measure the lifespan of large populations of cells and distinguish long- and short-lived mutants, all with minimal involvement of the experimenter. We also show that environmental pH is capable of significantly modulating lifespan depending on the growth media, highlighting how microfluidic technologies reveal determinants of lifespan that are otherwise difficult to ascertain.

cell biology↗

A genome-scale yeast library with inducible expression of individual genes

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships, including instances of feedback control. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which 5,687 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains Synthetic Genetic Array (SGA) screening markers and a unique molecular barcode, enabling high-throughput yeast genetics. We characterized YETI using quantitative growth phenotyping and pooled BAR-seq screens, and we used a YETI allele to characterize the regulon of ROF1, showing that it is a transcriptional repressor. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from other eukaryotic and prokaryotic systems shows that low native expression is a critical variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3EB42, that gives lower expression than Z3EV, a feature enabling both conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.

synthetic biology↗