Multiple-site genetic modifications in Escherichia coli using lambda-Red recombination and I-SceI cleavage.

Area of Science:

• Molecular Biology
• Microbial Genetics
• Synthetic Biology

Background:

• Bacterial genetic modifications are crucial for research and biotechnology.
• Existing methods for modifying bacterial chromosomes, such as single-locus plasmid systems in Escherichia coli, require improvement for simultaneous multiple-loci modifications.

Purpose of the Study:

• To develop an improved method for simultaneous multiple-loci modification in bacterial chromosomes.
• To enable efficient and concurrent genetic engineering at several genomic sites.

Main Methods:

• Generated an intermediate bacterial strain with resistance marker genes flanked by I-SceI recognition sites at multiple target loci.
• Transformed the intermediate strain with a donor plasmid containing desired alleles and a helper plasmid encoding λ-Red recombinase and I-SceI endonuclease.
• Utilized I-SceI-induced double-strand breaks (DSBs) and λ-Red-mediated recombination for genomic modifications.

Main Results:

• Successfully generated an intermediate strain suitable for multiplexed genetic engineering.
• Demonstrated the efficacy of the system in facilitating recombination between chromosomal DSBs and donor DNA fragments.
• Achieved simultaneous modifications at three different loci within the bacterial genome.

Conclusions:

• The developed method enables efficient simultaneous modification of multiple loci in bacterial chromosomes.
• This approach significantly advances the capabilities for bacterial genome engineering.