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

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. To efficiently transform DNA, we sought to harness TXTL as a distinct means to overcome DNA restriction. TXTL recapitulates transcription and translation in a lysate or solution of purified components, allowing the functional expression of RNA and protein from any bacterium in minutes to hours without the need for cell culturing or protein purification29. We envisioned a TXTL-based pipeline in which DNA MTases identified in a host bacterium (e.g. through bioinformatics or listed on the REBASE database30) are expressed and combined with DNA in a methylation reaction, and the resulting DNA is purified and transformed into the host. DNA lacking any methylation (e.g. PCR product, plasmid DNA extracted from a MTase-free strain) serves as the starting point to immediately circumvent Type IV R-M systems that cleave methylated DNA26. We call the resulting pipeline IMPRINT (Imitating Methylation Patterns Rapidly IN TXTL) (Fig. 1a). With IMPRINT, methylation by multiple MTases, including Type I MTases that also require expressing a specificity protein, is straightforward and completed in under a day. Any cytotoxicity concerns are also minimized due to the absence of replicating cells for plasmid methylation.