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KMID : 1094720210260061023
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Biotechnology and Bioprocess Engineering
2021 Volume.26 No. 6 p.1023 ~ p.1033
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Rapid and Efficient BAC Recombineering: Gain & Loss Screening System
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Kuk Myeong-Uk
Lee Yun-Haeng
Kim Jae-Won
Hwang Su-Young
Park Ji-Yun
Song Eun-Seon
Kwon Hyung-Wook
Oh Se-Kyung
Park Joon-Tae
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Abstract
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Recombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone. Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days. Overall, our new strategy may provide a new avenue for BAC recombineering, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.
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KEYWORD
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recombineering, lamda-red recombinase, gain & loss screening system
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