Recombineering in Non-Model Bacteria

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Recombineering, a method for in vivo genetic engineering, utilizes bacteriophage-encoded homologous recombination proteins.
• It allows precise genomic DNA modification at short homology sites (35-50 nt) without restriction enzyme dependence.
• The technology employs linear DNA substrates like PCR products, dsDNA, or ssDNA, introduced via electroporation.

Purpose of the Study:

• To review applications, challenges, and factors influencing ssDNA and dsDNA recombineering in various non-model bacteria.
• To highlight recent breakthroughs and new homologous recombineering systems from non-model bacteria.
• To provide guidance for researchers on selecting or developing recombineering systems for specific microbes.

Main Methods:

• Description of established phage λ Red and Rac RecET systems used in diverse bacteria.
• Introduction of novel homologous ssDNA and dsDNA recombineering systems from non-model bacteria.
• Detailed protocols for ssDNA recombineering in Shewanella species, including CRISPR/Cas9 as a counter-selection tool.

Main Results:

• Successful application of recombineering in a wide range of Gram-negative and Gram-positive non-model bacteria.
• Development and characterization of new ssDNA and dsDNA recombineering systems.
• Demonstration of CRISPR/Cas9 as an effective counter-selection method to enhance recombinant recovery.

Conclusions:

• Recombineering is a versatile tool for precise genome engineering in diverse bacterial species.
• New systems and strategies, including oligo-mediated recombination, expand the utility of recombineering in undomesticated bacteria.
• This work provides a decision-making framework to aid researchers in applying or developing recombineering strategies for their specific microbial targets.