Negative selection markers are extremely useful in genetic engineering as they enable scarless, marker-free manipulations, leading to cleaner phenotypes. Traditionally, most of these markers suffer from drawbacks like: low stringency, need for optimisation, and/or need for manipulation of the wild type strains. Furthermore, they are challenging to use in clinical strains. Our lab has recently developed a set of powerful negative selection tools, which overcomes these shortcomings and can be used in lab and clinical bacterial strains. The biggest improvement in our inducible toxin based system is that we have been able to achieve high stringencies (almost 100x higher than other negative selection markers), which are just 10 fold lower than those achieved by positive selection markers. This has been very useful in generating mutants and carrying out allelic exchange experiments in clinical strains of E. coli.
While carrying out these experiments we came across fluctuating background rates of false positive clones, ranging from spontaneous mutations/rearrangements within the negative selection module to large deletions around the cassette. The rate of excision of the module varied the most and was dependent on the region of insertion of the cassette in the genome; for example, pathogenicity islands and phages have dedicated excision mechanisms that could eliminate a negative selection cassette inserted into them. Although such excision is a potential problem for recombination, however, it also indicates variation in stability in different locations of the chromosome. This led to the idea that we could use our negative selection cassette to quantify chromosomal instability as a function of position across the entire genome. Our preliminary results have confirmed some previously known unstable regions and we are in pursuit of discovering novel regions which might be leading to genome plasticity in pathogenic and non-pathogenic strains of E. coli.