title: Quantifying Impact of Chromosome Copy Number on Recombination in Escherichia coli.
abstract: The ability to precisely and efficiently recombineer synthetic DNA into organisms of interest in a quantitative manner is a key requirement in genome engineering. Even though considerable effort has gone into the characterization of recombination in Escherichia coli, there is still substantial variability in reported recombination efficiencies. We hypothesized that this observed variability could, in part, be explained by the variability in chromosome copy number as well as the location of the replication forks relative to the recombination site. During rapid growth, E. coli cells may contain several pairs of open replication forks. While recombineered forks are resolving and segregating within the population, changes in apparent recombineering efficiency should be observed. In the case of dominant phenotypes, we predicted and then experimentally confirmed that the apparent recombination efficiency declined during recovery until complete segregation of recombineered and wild-type genomes had occurred. We observed the reverse trend for recessive phenotypes. The observed changes in apparent recombination efficiency were found to be in agreement with mathematical calculations based on our proposed mechanism. We also provide a model that can be used to estimate the total segregated recombination efficiency based on an initial efficiency and growth rate. These results emphasize the importance of employing quantitative strategies in the design of genome-scale engineering efforts.
authors.affiliation: Department of Chemical and Biological Engineering, 596 UCB, JSCBB, University of Colorado Boulder, Boulder, Colorado 80309, United States.
authors.forename: T Steele
authors.initials: TS
authors.lastname: Reynolds
authors.affiliation: Department of Chemical and Biological Engineering, 596 UCB, JSCBB, University of Colorado Boulder, Boulder, Colorado 80309, United States.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D011995:Recombination, Genetic
keywords: genome engineering; recombination efficiency