Book chapter · Journal article
Selection of Highly Expressed Gene Variants in Escherichia coli Using Translationally Coupled Antibiotic Selection Markers
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Microbial Evolution and Synthetic Biology, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
Stockholm University4
Strategies to select highly expressed variants of a protein coding sequence are usually based on trial-and-error approaches, which are time-consuming and expensive. We address this problem using translationally coupled antibiotic resistance markers. The system requires that the target gene can be fused at the 3'-end with a translational coupling element and an antibiotic resistance gene.
Highly expressed target genes can then be selected using a fast and simple whole cell survival assay in the presence of high antibiotic concentrations. Herein we show that the system can be used to select highly expressing clones from libraries sampling translation initiation sites.
Language: | English |
---|---|
Year: | 2018 |
Pages: | 259-268 |
Series: | Methods in Molecular Biology |
ISBN: | 1493972944 , 1493972952 , 9781493972944 and 9781493972951 |
ISSN: | 10643745 and 19406029 |
Types: | Book chapter and Journal article |
DOI: | 10.1007/978-1-4939-7295-1_16 |
ORCIDs: | Rennig, Maja and Nørholm, Morten H. H. |