Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing

TitleRecombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing
Publication TypeJournal Article
Year of Publication2013
AuthorsBoyle, NR, Reynolds, TS, Evans, R, Lynch, M, and Gill, RT
JournalBiotechnology Journal
Volume8
Issue5
Start Page515
Pagination515 - 522
Date Published05/2013
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.

DOI10.1002/biot.201200237
Short TitleBiotechnology Journal