title: Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing.
abstract: Recombineering has been an essential tool for genetic engineering in microbes for many years and has enabled faster, more efficient engineering than previous techniques. There have been numerous studies that focus on improving recombineering efficiency, which can be divided into three main areas: (i) optimizing the oligo used for recombineering to enhance replication fork annealing and limit proofreading; (ii) mechanisms to modify the replisome itself, enabling an increased rate of annealing; and (iii) multiplexing recombineering targets and automation. These efforts have increased the efficiency of recombineering several hundred-fold. One area that has received far less attention is the problem of multiple chromosomes, which effectively decrease efficiency on a chromosomal basis, resulting in more sectored colonies, which require longer outgrowth to obtain clonal populations. Herein, we describe the problem of multiple chromosomes, discuss calculations predicting how many generations are needed to obtain a pure colony, and how changes in experimental procedure or genetic background can minimize the effect of multiple chromosomes.
authors: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA.
authors: Nanette R
authors: NR
authors: Boyle
authors: T Steele
authors: TS
authors: Reynolds
authors: Ron
authors: R
authors: Evans
authors: Michael
authors: M
authors: Lynch
authors: Ryan T
authors: RT
authors: Gill
mesh_terms: D001709:Biotechnology; D004274:DNA, Recombinant / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods*; D011995:Recombination, Genetic
chemical_list: D004274:DNA, Recombinant