High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

Abstract

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis

“…Semi-planar PDMS-based drop makers have been designed to make small DE drops while avoiding full 3-D fabrication. ,, These devices use a planar lower channel surface and a step increase in channel height at the second junction which assists during surface treatment and DE formation. To form DEs in a planar device, the surface wetting of the channels must switch from hydrophobic to hydrophilic between the first and second junctions. ,, Previous efforts have demonstrated polymer deposition with flow confinement for the spatial patterning of the surface wetting of PDMS microchannels. ,, Although effective, polymer deposition methods are laborious to perform. The in-flow of the polymer treatment solution must be carefully balanced by a second fluid that blocks the polymer solution from entering the untreated portions of the device.…”

“…To form DEs in a planar device, the surface wetting of the channels must switch from hydrophobic to hydrophilic between the first and second junctions. 7,61,62 Previous efforts have demonstrated polymer deposition with flow confinement for the spatial patterning of the surface wetting of PDMS microchannels. 60,63,64 Although effective, polymer deposition methods are laborious to perform.…”

“…Droplet microfluidics has established itself for studying biological and biochemical systems with high throughput and low cost. − By forming millions of near-identical water in oil droplets, samples are partitioned enabling in-drop directed evolution, high throughput screening, , and single-molecule or single-cell analysis. − These foundational techniques rely upon the efficient compartmentalization enabled by monodisperse SE droplets, however, SE stability remains a persistent challenge . Despite the significant effort that has been devoted to developing surfactants that stabilize SE droplets during incubation, thermal cycling, reinjection, and other drop manipulations, droplet merging cannot be fully avoided. − Drop coalescence is known to vary with drop size , aqueous phase composition, − surfactant composition, , thermal gradients, , and shear stress .…”

“…Droplet microfluidics has established itself for studying biological and biochemical systems with high throughput and low cost. 1−4 By forming millions of near-identical water in oil droplets, samples are partitioned enabling in-drop directed evolution, 5 high throughput screening, 6,7 and single-molecule or single-cell analysis. 8−10 These foundational techniques rely upon the efficient compartmentalization enabled by monodisperse SE droplets, however, SE stability remains a persistent challenge.…”

Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis

“…Accordingly, spatially control of the surface wetting is required for DE formation using this approach. [44][45][46] Previous efforts have demonstrated polymer deposition for the spatial patterning of the surface wetting of PDMS microchannels. 47,48 Although effective, polymer deposition methods are laborious to perform as the in-flow of the polymer treatment solution must be carefully balanced by a second fluid that blocks the treatment solution from entering the untreated portions of the device.…”