Moss, Britney L, Lanctot, Amy, Ramos Báez, Román (March 2025) Testing AuxInYeast Synthetic Biology Strains via Fluorescence Flow Cytometry. Cold Spring Harbor Protocols. ISSN 1940-3402
Abstract
Understanding how the auxin hormone signaling pathway components come together to orchestrate cellular responses is key to engineering the growth and development of maize. Although a variety of techniques exist to measure auxin activities in plants, many are time- and resource-intensive or do not easily allow for high-throughput quantitative measurement of component libraries. The AuxInYeast system is a synthetic biology tool that facilitates complex biochemical analysis of the auxin hormone signaling pathway from essentially any plant. AuxInYeast uses Saccharomyces cerevisiae yeast as a heterologous expression platform for auxin signaling pathway components with fluorescent tags that facilitate measurement of auxin perception, repression, and activation. This protocol describes how to use fluorescence flow cytometry for these AuxInYeast experiments. As a case study, we focus on AuxInYeast strains built to measure maize auxin perception (i.e., those that express receptors and fluorescently tagged repressors that degrade upon auxin exposure). This protocol describes two different types of cytometry assays. The Steady-State Assay measures the extent of auxin-induced repressor degradation at one or two time points across many AuxInYeast strains and is particularly useful for initial assessment of whether auxin-induced degradation occurs and for dose response assays. The Time-Course Assay is used to measure auxin-induced repressor degradation dynamics over 2-3 h in a smaller number of strains. It is most useful for assessing the range of degradation rates across sets of repressors or receptors, and to precisely determine the impact of mutations and natural variation on degradation rate.
| Item Type: | Paper |
|---|---|
| Subjects: | Investigative techniques and equipment Investigative techniques and equipment > flow cytometry |
| CSHL Authors: | |
| Communities: | CSHL labs > Lippman lab |
| SWORD Depositor: | CSHL Elements |
| Depositing User: | CSHL Elements |
| Date: | 12 March 2025 |
| Date Deposited: | 14 Mar 2025 18:51 |
| Last Modified: | 14 Mar 2025 18:51 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/41818 |
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