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High-throughput direct screening of restriction endonuclease using microfluidic fluorescence-activated drop sorter based on SOS response in E. coli

Yizhe Zhang, Jeremy J. Agresti, Yu Zheng, View ORCID ProfileDavid A. Weitz
doi: https://doi.org/10.1101/2022.01.09.475563
Yizhe Zhang
1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA
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Jeremy J. Agresti
2John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA
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Yu Zheng
3New England BioLabs, Inc., Ipswich, MA 01938, USA
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David A. Weitz
2John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA
4Department of Physics, Harvard University, Cambridge, MA 02138, USA
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  • ORCID record for David A. Weitz
  • For correspondence: weitz@seas.harvard.edu
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ABSTRACT

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route for expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient fashion. Briefly, we construct a host bacterial cell to link the RE genotype to the phenotype of β-galactosidase expression based on the bacterial SOS response, and use a high-throughput microfluidic platform to isolate, detect and sort the REs. We employ this strategy to screen for the XbaI gene from constructed libraries of varied sizes. In single round of sorting, a 30-fold target enrichment was obtained within 1 h. The direct screening approach we propose shows potential for efficient search of desirable REs in natural samples compared to the conventional RE-screening method, and is amenable to being adapted to high-throughput screening of other genotoxic targets.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted January 11, 2022.
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High-throughput direct screening of restriction endonuclease using microfluidic fluorescence-activated drop sorter based on SOS response in E. coli
Yizhe Zhang, Jeremy J. Agresti, Yu Zheng, David A. Weitz
bioRxiv 2022.01.09.475563; doi: https://doi.org/10.1101/2022.01.09.475563
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High-throughput direct screening of restriction endonuclease using microfluidic fluorescence-activated drop sorter based on SOS response in E. coli
Yizhe Zhang, Jeremy J. Agresti, Yu Zheng, David A. Weitz
bioRxiv 2022.01.09.475563; doi: https://doi.org/10.1101/2022.01.09.475563

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