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. As a first demonstration, we MiCoded a set of synthetic leucine zippers (SYNZIPs) to allow an 8x8 matrix to be tested for specific interactions in a pool.  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.