Use of Streptomycin-Dependence as Selection/Counterselection for Sequential Oligonucleotide Mediated Recombineering in Mycobacteria

Oligonucleotide mediated recombineering is a powerful technique for genome editing in bacteria. In mycobacteria, researchers usually transform bacteria with two oligonucleotides: one conferring the mutation of interest, and a second conferring selectable antibiotic resistance. Multiple genetic modifications may be performed sequentially, however, this requires either the introduction of multiple antibiotic resistances or a tedious process of reversing antibiotic resistance between steps. Rather than using antibiotic resistance for selection, we instead develop a system that uses streptomycin-dependence. Because streptomycin-dependence can easily be selected both for and against, this allows a theoretically unlimited number of recombineering edits to be sequentially selected for, by toggling between streptomycin-sensitivity and dependence. After an even number of editing cycles, strains are automatically unmarked. Strains which have undergone an odd number of edits can be unmarked with a single additional recombineering step. This allows significantly easier exploration of combinatorial interactions between multiple mutations in mycobacteria.