A Broad Host Range Plasmid-Based Roadmap for ssDNA-Based Recombineering in Gram-Negative Bacteria.

Abstract

Recombineering is the use of phage recombination proteins to improve and facilitate bacterial genome engineering. Depending on the nature of the DNA template, double-stranded or single-stranded, the system needs three proteins (Gam, Exo, and Beta) or just one (Beta) to work properly. The use of this technique has been fundamental not only toward solving fundamental biological questions with reverse genetics but also for the generation of deep-engineered E. coli chassis strains. Unfortunately, the use of ssDNA recombineering is still limited to a narrow number of bacterial species. One of the reasons for that is the lack of proper recombinases to be efficiently used in different microorganisms and the lack of proper genetic tools to deliver and express this activity in a controlled way. Here, we describe a protocol to follow a simple workflow to identify, clone, and quantify the function of the selected recombinases in the organism of choice by cloning and expressing them in standardized broad host range plasmids. As an example of the method, we tested the use of the Ssr recombinase in P. putida EM42 by introducing a complete deletion of the target gene pyrF. The example shows how two parameters of the mutagenic oligo, i.e., length and phosphorothioate protection, affect thefinal outcome of the procedure.