High-throughput direct screening of restriction endonuclease using a microfluidic fluorescence-activated drop sorter based on the SOS response in Escherichia coli.
Analyst
; 149(13): 3575-3584, 2024 Jun 24.
Article
em En
| MEDLINE
| ID: mdl-38758107
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 to 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 manner. We constructed a host bacterial cell to link the genotype of REs to the phenotype of ß-galactosidase expression based on the bacterial SOS response, and used a high-throughput microfluidic platform to isolate, detect and sort the REs in microfluidic drops at a frequency of â¼800 drops per second. We employed this strategy to screen for the XbaI gene from the constructed libraries of varied sizes. In a single round of sorting, a 90-fold target enrichment was achieved within 1 h. Compared to conventional RE-screening methods, the direct screening approach that we propose excels at efficient search of desirable REs in natural samples - especially unculturable samples - and can be tailored to high-throughput screening of a wide range of genotoxic targets.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Resposta SOS em Genética
/
Enzimas de Restrição do DNA
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Escherichia coli
Idioma:
En
Revista:
Analyst
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos