In vivo capture system enabling rapid cloning of bacterial chromosomal segments.

Abstract

Homologous recombination-based genetic engineering, or recombineering, has greatly improved the precision and flexibility of bacterial genome manipulation. However, most existing approaches still require in vitro handling of target DNA, including PCR amplification, restriction digestion, or genomic DNA extraction prior to recombination. Here, an in vivo capture system enabling rapid cloning of bacterial chromosomal segments was developed, allowing direct recovery of genomic loci into plasmid vectors without prior amplification or in vitro DNA assembly. The system is based on λ Red-mediated homologous recombination between a linear PCR-amplified plasmid backbone and chromosomal loci in Gram-negative bacteria. Evaluation using the rcsA locus in Salmonella enterica revealed a minimal homology requirement for productive recombination. Homology arms of 10-20 bp supported plasmid recovery but failed to yield detectable on-target recombinants, whereas 30-40 bp arms reproducibly enabled accurate chromosomal segment capture. Under optimized conditions, recovered clones showed high on-target fidelity as confirmed by colony PCR and phenotypic analysis. Multiple independent recombinant plasmids were further validated by whole-plasmid sequencing, confirming precise junction formation and faithful recovery of the targeted chromosomal regions. Collectively, this study establishes a practical framework for an in vivo capture system for bacterial chromosomal segments and defines key parameters required for reliable recovery of accurate recombinant plasmids.