A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy
Name
A barcoding strategy.pdf
Size
850.27 KB
Format
Adobe PDF
Checksum (MD5)
ea2cb61cda1509c7050c62a73a7ab15e
Author(s)
Chen, Robert
Rishi, Harneet S.
Yamada, Masaki R.
Yeh, Vincent J.
Chow, Thomas
Cheung, Celia L.
Jones, Austin T.
Johnson, Terry D.
DeLoache, William C.
Dueber, John E.
Date Issued
July 2015
Journal
ACS Synthetic Biology
Publisher
American Chemical Society (ACS)
Citation
Chen, Robert et al. “A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.” ACS Synthetic Biology 4.11 (2015): 1205–1216.
Version
Author's final manuscript
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1021/acssynbio.5b00060
