Biotechnology and Bioprocess Engineering 2021; 26(6): 1023-1033  
Rapid and Efficient BAC Recombineering: Gain & Loss Screening System
Myeong Uk Kuk, Yun Haeng Lee, Jae Won Kim, Su Young Hwang, Ji Yun Park, Eun Seon Song, Hyung Wook Kwon, Sekyung Oh, and Joon Tae Park
Myeong Uk Kuk, Yun Haeng Lee, Jae Won Kim, Su Young Hwang, Ji Yun Park, Eun Seon Song, Hyung Wook Kwon*, Joon Tae Park*
Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Korea
Tel: +82-32-835-8090
E-mail: hwkwon@inu.ac.kr
Tel: +82-32-835-8841
E-mail: joontae.park@inu.ac.kr
Sekyung Oh*
Department of Medical Sciences, Catholic Kwandong University College of Medicine, Incheon 22711, Korea
Institute for Biomedical Research, Catholic Kwandong University International St. Mary’s Hospital, Incheon 22711, Korea
Tel: +82-10-6562-7585
E-mail: ohskjhmi@gmail.com
Received: December 8, 2020; Revised: February 2, 2021; Accepted: February 3, 2021; Published online: December 31, 2021.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
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.
Keywords: recombineering, lamda-red recombinase, gain & loss screening system


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