Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Novel High-Throughput DNA Part Characterization Technique for Synthetic Biology

  • Bak, Seong-Kun (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Seong, Wonjae (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Rha, Eugene (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Hyewon (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Seong Keun (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kwon, Kil Koang (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Haseong (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Lee, Seung-Goo (Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Received : 2022.07.07
  • Accepted : 2022.07.19
  • Published : 2022.08.28
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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 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.

Keywords

Acknowledgement

This research was supported by a grant from the Next-Generation BioGreen 21 Program (Grant No. PJ015808022022), Rural Development Administration, Republic of Korea, the Bio & Medical Technology Development Program (Grant No. 2021M3A9I4022731) of the National Research Foundation funded by the Ministry of Science and ICT of the Republic of Korea and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5402221).

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