There are many incentives to assess the function of essential genes in both laboratory and pathogenic strains of bacteria. Firstly, they serve as the starting point to enable minimal genome design to explain how some novel pathways may have evolved in earlier life. Secondly, understanding essential gene function may address the requirements to enhance and optimise biotechnological applications. Lastly, essential genes often encode key components in novel biochemical pathways, and these are often drug targets for antimicrobial developments.
Here, we present a novel two plasmid-based method that utilises the FRT-Flp system. Our strategy involves inserting an essential gene next to a traceable reporter, which is located between two unidirectional FRT sites (pFRT), and a second temperature sensitive plasmid which encodes a transcriptionally regulated Flp recombinase (pFlp). We have shown, using lambda recombinase, that our system can efficiently introduce deletion of chromosomal essential genes while retaining an FRT-flanked copy on a plasmid. Controlled Flp-cleavage offers the flexibility to assess conditions to bypass a lethal genetic defect.
We showcase this tool's versatility using two examples, including the nutritional bypass for the loss of Ketoacyl-ACP synthase I and the assessment of mutagenised LptE, an essential lipopolysaccharide transporter protein, with unique properties compared to the wild-type variant.