A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy
| Autor: | Thomas Chow, Vincent J. Yeh, Vladimir Potapov, Terry D. Johnson, Masaki R. Yamada, Harneet S. Rishi, Celia L. Cheung, Robert Chen, William C. DeLoache, John E. Dueber, Amy E. Keating, Austin T. Jones |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Genetics
Leucine zipper Electronic Data Processing Microscopy Biomedical Engineering Proteins General Medicine Computational biology Biology Barcode Subcellular localization Biochemistry Genetics and Molecular Biology (miscellaneous) Phenotype Article law.invention High-Throughput Screening Assays Synthetic biology law Synthetic Biology Throughput (business) |
| Popis: | Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms. |
| Databáze: | OpenAIRE |
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