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Updated: May 16, 2025

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
Published on: July 7, 2020
Development of a Transposon-Based Genome Engineering Toolkit for Efficient and Adaptable Genetic Modifications in
Seungwoo Baek1, Bogun Kim1, Duleepa Pathiraja2
1Department of Biotechnology, School of Life Science and Biotechnology, Korea University, Seoul 02847, Republic of Korea.
Abstract:
Advances in genome engineering of fungal strains are rapidly progressing, driven by the increasing interest in fungal biotechnology. Given the unique genomic and cellular complexity of fungi, each strain benefits from a tailored toolkit for efficient genome engineering. Here, we present a transposon-based engineering toolkit specifically optimized for Wolfiporia cocos, a species valued for its bioactive compounds. This toolkit significantly improves transformation efficiency, enabling multiplexed gene integration and facilitating rapid, flexible prototyping by assembling multiple genes into transposomes in a cocktail format, which bypasses the need for an intricate genetic circuit assembly. Engineered strains demonstrated stable expression across generations, as confirmed by successful genomic integration. Additionally, we identified six native W. cocos promoters from transcriptomic data, with two showing robust, constitutive expression in the mycelium of engineered strains. This transposon-based toolkit offers a versatile resource for synthetic biology, supporting efficient and adaptable genetic modifications within fungal systems.
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