Impact of Recovery Time On Recombination Efficiency in Escherichia Coli

With the rapid advancement of DNA synthesis technologies, the ability to efficiently recombineer user-defined DNA into organisms of interest has become increasingly important. Recombination efficiency in Escherichia coli has been the subject of many studies. Despite these efforts, there is still considerable variability in reported recombination efficiencies. We hypothesized that multiple chromosome copies within E. coli could partially explain reported variability. With more than one chromosomal target per E. coli cell, population phenotype expression levels could change as recombineered chromosomes are segregated to daughter cells. A simple predictive tool was developed to estimate cell population phenotype and genotype during outgrowth after recombination. We found that recombineered chromosomes were increasingly segregated within the population; leading to a dilution of the phenotype of interest but an increase in chromosomal homogeneity.  These results were confirmed experimentally leading to the conclusion that recovery time should be accounted for when reporting recombineering efficiencies.