A novel approach to isolate unselected recombinants facilitates markerless DNA recombineering

Abstract

Current screening methods for unselected recombinants, such as colony hybridization, are essentially in situ approaches. Using the “founder effect” (Mayr E., 1963), a classical population genetics principle, we developed an en masse screening procedure in liquid cultures to isolate markerless recombinants generated by recombineering. The founder effect states that allele frequency can be randomly increased by establishing a new sub‐population from a larger population. Thus, we sampled an E. coli culture so that it contained only a few recombinants and subdivided it into 96 sub‐cultures by plating in LB plates. Recombinant‐positive wells were detected by PCR using a multi‐channel electronic pipetter. Each positive well was founded by at least 1 recombinant cell, but the number of total cells decreased by almost 100‐fold. This implies that, if there is no selection against DNA recombinants and the DNA modification is stable, approximately 100‐fold enrichment can be achieved at every sampling/dilution cycle. Using PCR screening, we isolated pure recombinant clones containing a BAC deletion and two separate DNA replacements either from plated clones after the second cycle (max 104‐fold enrichment, 7 days) or from liquid cultures after the third cycle (max 106‐fold enrichment, 10 days). These results demonstrate that our novel approach to isolate unselected recombinants facilitates markerless DNA recombineering.