Engineering Genetically Intractable Bacteria: Targeted elimination of restriction-modification motifs in plasmid sequences with the SyngenicDNA Tool Generator (SyToGen)

• DOI: 10.52843/cassyni.s1m8kg
• Publication Date: 2024-09-13T13:20:08Z
• Authors: Gianmarco Piccinno

The vast majority of bacteria that can be grown in a laboratory remain genetically intractable, beyond the power of genetics for elucidating function or engineering for human use. Inherent diversity of genetic defenses, primarily Restriction-Modification (RM) systems, across bacterial species and individual strains remains a fundamental barrier to human-made DNA constructs during genetic engineering of bacteria. Previously, we described an approach to evade RM systems through the creation of SyngenicDNA; de-novo synthesized DNA sequences that are precisely altered to achieve epigenetic compatibility with a desired bacterial host and to prevent RM defense activation upon artificial transformation. SyngenicDNA-based genetic tools have increased transformation efficiencies by several orders of magnitude, yet still require manual ad-hoc sequence alterations for their generation. To remove the need for manual introduction of site-specific DNA alterations in silico, and to improve the accessibility, speed of generation, and reproducibility of SyngenicDNA, we introduce the SyngenicDNA Tool Generator (SyToGen). SyToGen is a computational workbench designed to assist users who work with bacteria that are currently genetically intractable. The software adheres to the manual workflow for generating SyngenicDNA, namely target identification, in silico tool assembly and sequence adaptation, but rapidly and reproducibly provides users with RM-silent DNA templates for de novo synthesis and assembly. SyToGen output plasmid sequences retain the form and functionality of the original genetic tool, but are now uniquely re-designed at the DNA level to bypass RM barriers and operate within the specific bacterium of interest.