title: A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.
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.
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Harneet S
authors.initials: HS
authors.lastname: Rishi
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Potapov
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Masaki R
authors.initials: MR
authors.lastname: Yamada
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Yeh
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Chow
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Celia L
authors.initials: CL
authors.lastname: Cheung
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Austin T
authors.initials: AT
authors.lastname: Jones
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Terry D
authors.initials: TD
authors.lastname: Johnson
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Keating
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: William C
authors.initials: WC
authors.lastname: DeLoache
authors.affiliation: Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.|Department of Biology, and #Department of Biological Engineering, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: John E
authors.initials: JE
authors.lastname: Dueber
mesh_terms: D001330:Electronic Data Processing; D057166:High-Throughput Screening Assays / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D011506:Proteins / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D011506:Proteins
keywords: coiled-coil zippers; library screening; microscopy barcodes; synthetic biology