(429c) A Cell-Free Pipeline for Rapidly Imprinting Complex Methylation Patterns on DNA to Enhance Plasmid Transformation in Bacteria (Industry Candidate)

Genetic tools are critical to study and engineer a diverse assortment of bacteria for next-generation industrial biocatalysts or as cell-based diagnostics and therapeutics. However such tools are utterly lacking in the vast majority of bacteria, with inefficient DNA transformation as one of the prevailing culprits. A principal barrier to DNA transformation is host restriction-modification (R-M) systems that produce a unique and often complex methylation pattern on host DNA and degrade incoming DNA lacking this pattern. Reproducing the host’s methylation pattern on a transformable shuttle plasmid has been shown to evade host R-M systems, although all available methods require cellular expression of every methyltransferase thus are extremely laborious and regularly impeded by methyltransferase-dependent cytotoxicity. Here, we describe an alternative approach where every methyltransferase gene is expressed via cell-free transcription-translation (TXTL) to rapidly reproduce and imprint complex methylation patterns upon a plasmid to enhance DNA transformation. We call this method IMPRINT (Imitating Methylation Patterns Rapidly IN TXTL). We first showed that the well-characterized Dam and Dcm methyltransferases from E. coli could be efficiently expressed and methylate supplied DNA, thus protecting the DNA from restriction digestion, while the TXTL reaction did not imbue any methylation on its own. We then applied and further optimized IMPRINT to a pathogenic strain of Salmonella enterica, where we identified which combination of host methyltransferases are needed to maximize DNA transformation. We are now implementing this approach in a set of probiotic Lactobacilli strains with varying R-M systems to explore strain-dependent barriers to DNA transformation and how DNA transformation can be consistently achieved across strains. Finally, we are applying this method to promising probiotic species that have been previously understudied due to extremely poor or no prior DNA transformation ability. Overall, IMPRINT represents a streamlined pipeline to rapidly boost DNA transformation efficiency across the rich diversity of bacteria by evading prominent yet strain-specific defense systems. This approach thus should help others access a much wider range of bacteria for molecular and synthetic biology experiments, in turn broadening our understanding of bacterial genotype-phenotype relationships and driving more effective bacterial engineering solutions.