Journal
BIOTECHNOLOGY JOURNAL
Volume 8, Issue 5, Pages 515-522Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201200237
Keywords
Genome engineering; Lambda Red; Multiple chromosomes; Rec E/T; Recombineering efficiency
Funding
- OPX Biotechnologies Inc.
Ask authors/readers for more resources
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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available