The endogenous RecA homology-based recombination system has been typically used to edit the bacterial chromosome in Gram-negative bacteria, and although this technique is efficient, it is also time-consuming. An alternative to this is the use of recombineering, which is based on the use of recombination proteins derived from bacteriophages that stimulate the recombination of incoming DNA within the native chromosome. This technology, that was first developed for Escherichia coli and then for other bacterial species (including Lactococcus lactis, Lactobacillus reuteri and Pseudomonas syringae, among others), can be used for simple gene deletions, but also for deep genome engineering. Nevertheless, recombineering has a limitation: it depends on the use of an appropriate recombinase to mediate the genome editing process, and this knowledge is limited to a small number of bacterial species.
In this context, this research focuses on developing a simple workflow to identify, clone and quantify the function of recombinases in selected organisms. With this approach, users can: (1) screen and test the efficiency of different recombinases in Gram-negative bacterial species; (2) test the minimum length of ssDNA oligo required to achieve efficient recombination; and (3) test the effect of phosphorothioate-protected oligonucleotides on the overall efficiency.