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A cell-free pipeline for recreating methylation patterns radically enhances DNA transformation in bacteria

ABSTRACT: The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic-biology chasses. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here we report IMPRINT, a generalized, rapid and scalable approach to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes cell-free systems to express DNA methyltransferases from the bacterial host’s restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the host DNA’s methylation pattern, circumventing restriction and enhancing transformation. Unlike established approaches, IMPRINT can be completed in under one day, readily accommodates all methyltransferase types, and avoids methylation-induced cytotoxicity. With IMPRINT, we efficiently multiplex DNA methylation and maximize plasmid transformation in gram-negative and gram-positive bacteria. We also developed a high-throughput pipeline that identifies the most consequential methyltransferases in one transformation. Overall, IMPRINT can vastly enhance DNA transformation, enabling use of increasingly sophisticated genetic manipulation tools across the bacterial world.

ORGANISM(S): Bifidobacterium breve Escherichia coli

PROVIDER: GSE240651 | GEO | 2024/04/07

REPOSITORIES: GEO

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