Published: April 18th, 2021
Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.
Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105 Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains' average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.
Growth phenotypes contribute critically to yeast fitness. Natural selection can efficiently distinguish between lineages with growth rates differing by the inverse of the effective population size, which can exceed 108 individuals1. Furthermore, variability of growth rates among individuals within a population is an evolutionarily relevant parameter, as it can serve as the basis for survival strategies such as bet hedging2,3,4,5,6. Therefore, assays that allow for highly a....
1. Preparation of Randomized Plates (Prior to Experiment Day)
The novelty of this protocol is that growth rate can be calculated for individual cells within a population by tracking their growth into microcolonies through time-lapse imaging (Figure 2A). Because microcolonies grow for many hours in a planar manner due to the presence of concanavalin A, their areas can be tracked throughout the experiment, and a linear fit to the change in the natural log of the area over time can be used to calculate growth rate for each individual colony observed
The protocol described here is a versatile assay that allows cell growth and gene expression to be monitored simultaneously at the level of individual microcolonies. Combining these two modalities yields unique biological insights. For example, previous work has used this assay to show a negative correlation between expression of the TSL1 gene and microcolony growth rate in isogenic wildtype cells by measuring both simultaneously7,10. It is also possible.......
We thank Naomi Ziv, Sasha Levy and Shuang Li for their contributions to developing this protocol, David Gresham for shared equipment, and Marissa Knoll for help with video production. This work was supported by National Institutes of Health grant R35GM118170.....
|500 mL Bottletop Filter .22 µm PES Sterilizing, Low Protein Binding, w/45mm Neck
|used to filter the media
|BD Falcon*Tissue Culture Plates, microtest u-bottom
|96-well culture tubes used to freeze cells, pre-grow cells, and dilutions
|BD Syringes without Needle, 50 mL
|Used to filter the Concanavalin A
|Costar Sterile Disposable Reagent Reservoirs
|reagent reservoirs used to pipette solutions with multichannel pipette
|Costar Thermowell Aluminum Sealing Tape
|96-well plate seal for pre-growth and freezing
|lint and static free Kimwipes
|lint and static free wipes to keep microscope plate bottom free of debris and scratches
|Nalgene Syringe Filters
|0.2 μm pore size, 25 mm diameter; used to filter concanavalin A solution
|Minimal chemically defined media (MD; 2% glucose)
|alternative microscopy media used for yeast pre-growth and growth during microscopy
|Synthetic Complete Media (SC; 2% glucose)
|microscopy media used for yeast pre-growth and growth during microscopy
|Yeast extract-peptone-dextrose (YEPD; 2% glucose) medium
|cell growth prior to freezing down randomized plates
|Breathe-Easy sealing membrane
|breathable membranes used to seal plate during microscopy experiment. At this stage breathable membranes are reccomended because they prevent condensation in the wells and allow for better microscopy images
|Brooks 96-well flat clear glass bottom microscope plate
|Concanavalin A from canavalia ensiformis (Jack Bean), lyophilized powder
|Make 5x concanavalin A solution and freeze 5ml of 5x concanavalin A in 50 mL conical tubes at -80 °C
|MAH.5, MAH.96, MAH.52, MAH.66, MAH.11, MAH.58, MAH.135, MAH.15, MAH.44, MAH.132
|Haploid mutation accumulation strains in a laboratory background, described in Hall and Joseph 2010
|Progeny of the ancestral Hall and Joseph 2010 mutation accumulation strain, transformed with YFR054cΔ::Scw11P::GFP
|Misonix Sonicator S-4000 with 96-pin attachment
|Nikon Eclipse Ti-E with Perfect Focus System
|Inverted microscope with automated stage and autofocus system
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