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Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

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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.

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Abbreviations

BAC:

Bacterial artificial chromosome

GOI:

gene-of-interest

HR:

homology region

KanR :

kanamycin resistant gene

CamR :

chloramphenicol resistant gene

AmpR :

ampicillin resistant gene

NeoR :

neomycin resistance gene

Kan:

kanamycin

Cam:

chloramphenicol

Amp:

ampicillin

BTV:

BAC targeting vector

BTC:

BAC targeting cassette

CTV:

Cam targeting vector

CTC:

Cam targeting cassette

Fwd:

forward

Rev:

reverse

FLP :

flippase

SOC:

Super Optimal broth with Catabolite repression

DW:

Distilled Water

OD:

optimal density

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Acknowledgements

This research was supported by priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A0304195411). This research was also supported by Research Assistance Program (2019) in the Incheon National University.

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Authors and Affiliations

  1. Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, 22012, Korea

    Myeong Uk Kuk, Yun Haeng Lee, Jae Won Kim, Su Young Hwang, Ji Yun Park, Eun Seon Song, Hyung Wook Kwon & Joon Tae Park

  2. Department of Medical Sciences, Catholic Kwandong University College of Medicine, Incheon, 22711, Korea

    Sekyung Oh

  3. Institute for Biomedical Research, Catholic Kwandong University International St. Mary’s Hospital, Incheon, 22711, Korea

    Sekyung Oh

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  1. Myeong Uk Kuk
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  2. Yun Haeng Lee
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  7. Hyung Wook Kwon
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Contributions

MUK, KHW, SO and JTP conceived of and designed the experiments. MUK, YHL, JWK and SYH performed the experiments. MUK, KHW, SO and JTP wrote and edited the paper. All authors have read and approved the manuscript

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Correspondence to Hyung Wook Kwon, Sekyung Oh or Joon Tae Park.

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Conflict of Interest The authors declare no competing financial interests.

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Kuk, M.U., Lee, Y.H., Kim, J.W. et al. Rapid and Efficient BAC Recombineering: Gain & Loss Screening System. Biotechnol Bioproc E 26, 1023–1033 (2021). https://doi.org/10.1007/s12257-020-0382-1

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  • DOI: https://doi.org/10.1007/s12257-020-0382-1

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