A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy

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.