Novel High-Throughput DNA Part Characterization Technique for Synthetic Biology

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 intensiti...

Full description

Saved in:
Bibliographic Details
Published in:Journal of microbiology and biotechnology 2022-08, Vol.32 (8), p.1026-1033
Main Authors: Bak, Seong-Kun, Seong, Wonjae, Rha, Eugene, Lee, Hyewon, Kim, Seong Keun, Kwon, Kil Koang, Kim, Haseong, Lee, Seung-Goo
Format: Article
Language:Korean
Subjects:
circuit design
DNA parts
image analysis
long-read sequencing
Synthetic biology
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1017-7825
1738-8872