High-throughput Generation and Evaluation of Genetic Circuit Libraries

Haseong Kim^1),2),4) Byung-Kwan Cho^1),3) Seong-Goo Lee^1),2),4)

¹⁾K-Biofoundry^β, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea

²⁾Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea

³⁾Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

⁴⁾Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, Republic of Korea

This study presents a high-throughput part library characterization technique by using solid plate-based quantitative fluorescence assay for phenotyping, and barcode tagging-based long-read sequencing for genotyping. High concentration of DNA parts in the form of duplex oligo were directly used for part library assembly which can generate diverse phenotypes. The barcode tagging-based long-read sequencing technique enabled rapid identification of all DNA part combinations with a single sequencing experiment. 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. Using this techniques, forty-four DNA parts (21 promoters and 23 RBSs) were successfully characterized in 72 h. K-biofoundry beta is building a part repository, Partbank, for storing the quantitatively characterized parts and providing a circuit design software tool. We anticipate that this high-throughput technique with automated machines will contribute to increase the speed and scale in optimizing genetic circuits and metabolic pathways.