I-PREFR: Inverse PCR-Based Restriction Enzyme FRee Unidirectional Strategy for Rapid Markerless Chromosomal Gene Deletion and Reconstitution in Bacteria Using Suicide Vectors.

Abstract

The standard protocols for allelic exchange using homologous recombination deploy suicide vectors with negative selection markers. However, the use of multiple restriction enzymes to generate sticky ends in the vector and the insert for cloning is time-consuming, resource-intensive, and challenging. The advent of next-generation proofreading enzymes is enabling researchers to routinely carry out long-range PCR. Hence, amplifying 5-6 kb of complete low-complex DNA cloning vectors and 2-3 kb of complex genomic regions is much easier. Here, we report a simple, accurate, rapid, and unidirectional approach for chromosomal in-frame gene deletion and complementation by reconstitution of the full-length gene without using any restriction enzymes. The method requires long-range PCR using Phusion polymerase to linearize the vector and amplify the target gene to create a recombinant vector (pRM1) and further inverse PCR amplification of pRM1 to create a recombinant vector (pRM4) with a deleted version of the gene. The cloning steps involve the use of kinase and ligase for phosphorylation and ligation steps, respectively. The recombinant plasmid, pRM4, is finally transformed into electrocompetent cells of Xanthomonas sontii, a gram-negative phytobacterium, for final genomic integration/excision to obtain an in-frame gene deletion mutant (PPL1RM15). Gene reconstitution for complementation is carried out by electroporating the deletion mutant with the recombinant plasmid (pRM1) carrying the wild-type allele. Clean gene mutation, allele restoration, and plasmid excision are confirmed using whole-genome sequencing. Key features • The protocol is cost-effective and simple, eliminating the need for restriction enzymes and multiple sets of lengthy primers with restriction sites. • The protocol requires only kinase, ligase, and polymerase, along with three sets of standard-sized desalted primers. • The protocol is unidirectional; no need to create two different recombinant plasmids for gene deletion and complementation by reconstitution of the full-length gene. • The difference in size between empty and recombinant vectors facilitates the easy screening of transformed Escherichia coli colonies through colony PCR. • The strategy can be applied to any bacteria using a suitable suicide vector with appropriate positive and negative selection markers.