A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria.
Mol Cell
; 84(14): 2785-2796.e4, 2024 Jul 25.
Article
em En
| MEDLINE
| ID: mdl-38936361
ABSTRACT
The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.
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Base de dados:
MEDLINE
Assunto principal:
Transcrição Gênica
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Biossíntese de Proteínas
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Sistema Livre de Células
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Metilação de DNA
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article