Minicircle knockout

This work describes high efficiency transformation and vector-free allelic exchange knockout in non-model fish-pathogenic bacterium Photobacterium damselae subsp. piscicida (Pdp). This mutagenesis approach is simple, broadly-applicable, has low off-target mutation rate, and is also efficient for targets located on a small plasmids.Pdp is a member of the Vibrionaceae , which was thought to be poorly transformable after evaluation of multiple techniques for competent cell preparation in 1995. Thus, transformation could not have been used to deliver the allelic exchange DNA in knockout mutagenesis experiments. Instead, conjugative transfer of pir/RP4 suicide vectors from SM10/S17-1 lambda pir donor strains was employed, despite high off-target mutation rate reported multiple studies. Here we have achieved high efficiency transformation in Pdp using a single-ingredient buffer, a salt-free highly concentrated sucrose solution. This method is used in phylogenetically distant hard-to-transform streptococci, and it is likely to be efficient in a wide range of bacteria, including species and strains recalcitrant to other competent cell preparation techniques.We have also highly simplified the constructs required for making knock-outs. Ultimately, the only DNA sequences essential for allelic exchange mutagenesis are 0.5-1.5 kB sequences flanking the target gene (upstream and downstream homology arms) and selection marker/s, which are spliced to form an allelic exchange construct (AEC). Thus, where high efficiency transformation is available, AEC can be delivered directly and there should be no need to clone it into a plasmid vector. Here we deliver AEC in a form of a knockout minicircles, minimalistic AEC generated and circularised by Gibson Assembly. Since plasmid backbones often contain ubiquitous, unstable, and repetitive sequences, chances of off-target variants are reduced if they can be omitted and do not integrate into host's DNA along with AEC. In addition, minicircle approach can improve mutagenesis of genes located on small plasmids whose stability may be compromised by integration of suicide vector backbone. Sequencing of clones generated during knockout mutagenesis of the aip56 gene has confirmed that competent cell preparation using sucrose treatment and electroporation/integration of minicircles have no off-target pro-mutagenic effects. In contract, random large deletions were found in most clones subjected to sacB negative selection. It appears that prolonged exposure to high sucrose concentrations has activated transposable elements (TE) movement , and, consequently, sacB may be sub-optimal counter-selectable marker for genetic modification of bacteria with TE-rich genomes.