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KMID : 0545120220320081026
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Journal of Microbiology and Biotechnology
2022 Volume.32 No. 8 p.1026 ~ p.1033
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Novel High-Throughput DNA Part Characterization Technique for Synthetic Biology
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Bak Seong-Kun
Seong Won-Jae
Rha Eu-Gene
Lee Hye-Won
Kim Seong-Keun
Kwon Kil-Koang
Kim Ha-Seong
Lee Seung-Goo
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Abstract
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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 the fluorescence intensities of colonies on plates were comparable to fluorescence at the single-cell level from a high-end, flow-cytometry device and developed a 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 and be useful for building genetic circuits and metabolic pathways in synthetic biology.
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KEYWORD
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Synthetic biology, DNA parts, circuit design, long-read sequencing, image analysis
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