Phenotypic Patterning through Copy Number Adaptation to Environmental Gradients.

We recently described a paradigm for engineering bacterial adaptation using plasmids coupled to the same origin of replication. In this study, we use plasmid coupling to generate spatially separated and phenotypically distinct populations in response to heterogeneous environments. Using a custom microfluidic device, we continuously tracked engineered populations along induced gradients, enabling an in-depth analysis of the spatiotemporal dynamics of plasmid coupling. Our observations reveal a pronounced phenotypic separation within 4 h exposure to an opposing gradient of AHL and arabinose. Additionally, by modulating the burden strength balance between coupled plasmids, we demonstrate the inherent limitations and tunability of this system. Intriguingly, phenotypic separation persists for an extended time, hinting at a biophysical spatial retention mechanism reminiscent of natural speciation processes. Complementing our experimental data, mathematical models provide invaluable insights into the underlying mechanisms and guide optimization of plasmid coupling for prospective applications of environmental copy number adaptation engineering across separated domains.

Fabrication of Microfluidic Devices for Continuously Monitoring Yeast Aging.

For several decades, aging in Saccharomyces cerevisiae has been studied in hopes of understanding its causes and identifying conserved pathways that also drive aging in multicellular eukaryotes. While the short lifespan and unicellular nature of budding yeast has allowed its aging process to be observed by dissecting mother cells away from daughter cells under a microscope, this technique does not allow continuous, high-resolution, and high-throughput studies to be performed. Here, we present a protocol for constructing microfluidic devices for studying yeast aging that are free from these limitations. Our approach uses multilayer photolithography and soft lithography with polydimethylsiloxane (PDMS) to construct microfluidic devices with distinct single-cell trapping regions as well as channels for supplying media and removing recently born daughter cells. By doing so, aging yeast cells can be imaged at scale for the entirety of their lifespans, and the dynamics of molecular processes within single cells can be simultaneously tracked using fluorescence microscopy. Key features This protocol requires access to a photolithography lab in a cleanroom facility. Photolithography process for patterning photoresist on silicon wafers with multiple different feature heights. Soft lithography process for making PDMS microfluidic devices from silicon wafer templates.