Novel high-throughput DNA part characterization technique for synthetic biology

Abstract

This study presents a novel DNA part characterization technique that increases throughput by combinatorial DNA part assembly, solid plate-based quantitative fluorescence assay for phenotyping, and barcode tagging-based long-read sequencing for genotyping. We confirmed that fluorescence intensities of colonies on plates were comparable to single-cell level fluorescence from a high-end flow-cytometry device and developed high-throughput image analysis pipeline. The barcode tagging-based long-read sequencing technique enabled rapid identification of all DNA parts and their combinations with a single sequencing experiment. Using our techniques, forty-four DNA parts (21 promoters and 23 RBSs) were successfully characterized in 72 h without any automated equipment. We anticipate that this high-throughput and easy-to-use part characterization technique will contribute to increasing part diversity that are useful for building genetic circuits and metabolic pathways in synthetic biology.