Abstract
ABSTRACTThe 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 based on cell-free transcription-translation (TXTL) systems to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from the bacterial host’s restriction-modification systems. The expressed methyltransferases then methylate DNAin vitroto match the host DNA’s 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 developed a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to facilitate a library screen for translational rules in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling use of increasingly sophisticated genetic manipulation tools across the bacterial world.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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