Scarless One-Tube Genome Assembly via Computationally Optimized Uracil-DNA Glycosylase Reactions

Abstract

Synthetic biology enables the creation of systems such as bacteriophage (phage)-based biosensors, leveraging the innate specificity and efficiency of phages to rapidly identify pathogens. However, the current genome assembly and editing methods, including Gibson Assembly, Golden Gate Assembly, and CRISPR-Cas systems, have limitations that can hinder speed and flexibility, especially when complex modifications are needed. This study introduces a novel means for generating engineered bacteriophages through a one-pot, modular in vitro genome assembly platform utilizing uracil-DNA glycosylase, which allows genome modification without requiring extended overlaps, the removal of restriction enzyme sites, a Cas system, or homologous recombination. The design also minimizes the risk of secondary structure formation (e.g., hairpins), allowing for a more efficient assembly of fragments. To demonstrate functional genome engineering, we incorporated a NanoLuc luciferase reporter gene into the T7 genome, producing a recombinant phage capable of detecting E. coli, a strategy consistent with our previous work on waterborne pathogen detection. This platform enables rapid and flexible synthetic genome construction with high functional assembly efficiency, with broad applications in phage engineering, biosensing, and synthetic biology.